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Calprotectin consists of the Ca2+-binding proteins S100a8 and S100a9 that are induced in epithelial cells in response to tissue damage and infection. Both proteins are also secreted by activated innate immune cells and numerous studies demonstrate their crucial role in pathological conditions of acute and chronic inflammation.
Here, we established a conditional mouse model with simultaneous S100a8 and S100a9 transgene expression in hepatocytes (TgS100a8a9hep) under the control of doxycycline to unravel the role of epithelial-derived Calprotectin on tissue homeostasis and inflammation. TgS100a8a9hep mice displayed a significant enrichment of neutrophils in peripheral blood and tissues with high blood content. Interestingly, Cxcl1 transcription was significantly induced in the liver of TgS100a8a9hep mice and primary hepatocytes derived thereof as compared to Control mice, accompanied by an increase of Cxcl1 serum levels. However, expression of other chemokines with a known function in neutrophil mobilization from the bone marrow, e.g. Csf3 and Cxcl2, was not altered. Doxycycline treatment of TgS100a8a9hep mice reduced Cxcl1 expression in the liver and resulted in normal numbers of neutrophils.
In summary, our data demonstrate for the first time that hepatocyte-specific S100a8 and S100a9 expression induces a systemic mobilization of neutrophils by a specific activation of Cxcl1 transcription in the liver.
Keywords:Calgranulins; Calprotectin; Hepatocytes; Neutrophils; Chemokines; Immune cell mobilization
S100 proteins are a family of small Ca2+-binding proteins [1] which share a broad spectrum of functions including regulation of enzyme activity, Ca2+-homeostasis, and interaction with components of the cytoskeleton. Belonging to this family, S100A8 and S100A9 also known as migration inhibitory factor-related protein 8 (MRP8) and 14 (MRP14) are low molecular weight proteins of 8 kDa and 14 kDa, respectively, and were initially described as proteins expressed and released by infiltrating phagocytes [2,3]. S100A8 and S100A9 form preferably heterodimers also known as Calprotectin (S100A8/A9) whereas monomers and homodimers could not be detected in isolated granulocytes and monocytes [4]. Although expression of S100A8/A9 under physiologic conditions is restricted to cells of myeloid origin including circulating neutrophils, monocytes, and eosinophils [5,6], strong induction of S100A8/A9 was detected in epithelial and endothelial cells under inflammatory and stress conditions [7]. Furthermore, marked expression of S100A8/A9 was found in many tumors of epithelial origin including lung, breast, gastric, colorectal and prostate cancer [8]. These data strongly imply important functions of S100A8/A9 under conditions of tissue damage and inflammation. Accordingly S100A8/A9 is strongly induced in a broad variety of different chronic inflammatory disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, arthritis, and psoriasis [8]. S100A8/A9 is able to support an inflammatory response via both intra- and extracellular mechanisms. In fact, intracellular S100A8/A9 has been shown to facilitate leukocyte trafficking through the transendothelial membrane via microtubule reorganization [9], upregulation of CD11b [10], as well as increase in the binding affinity of CD11b to the endothelial expressed integrin ICAM-1 [11]. Moreover S100A8/A9 is able to boost the inflammatory response by promotion of NADPH oxidase activity and production of ROS in phagocytes [12] and epithelial cells [13]. After secretion at the site of insult by epithelial and innate immune cells, S100A8/A9 directly acts as a chemoattractant for leukocytes, as demonstrated both in vitro and in vivo[14]. Importantly, S100A8/A9 is known to amplify pro-inflammatory signals, as it is able to induce the production and release of many cytokines and chemokines by epithelial and innate immune cells, e.g. CXCL1, CXCL2, IL-6, and TNF [15]. In this context S100A8/A9 was shown to promote pre-metastatic niche formation in the lungs of tumor-bearing mice [16,17]. Moreover, S100A8/A9 can promote its own expression by a feed-forward loop [15].
In vivo studies highlighted a crucial role for S100a8/a9 in the promotion of inflammation [18] and inflammation-induced carcinogenesis [19] by using S100a9-/- animals in models of antigen-induced arthritis and colon carcinogenesis, respectively. However these studies focused on the role of myeloid-derived S100a8/a9. In contrast, no causal link between epithelial-derived S100a8/a9 and the establishment of chronic inflammation and subsequent tumor-development has been demonstrated yet. Strong experimental evidence indicates that S100A8/A9 overexpression in hepatocytes is associated with liver carcinogenesis as epidemiological studies showed a profound correlation of both proteins with high grade hepatocellular carcinoma in humans [20]. Moreover, our group demonstrated a strong increase in S100a8/a9 in the Mdr2-/- model of inflammation-induced carcinogenesis in a NF-κB dependent manner [21]. Interestingly, elevated S100a8/a9 levels protected hepatocellular carcinoma cell lines from TNF-mediated apoptosis and promoted the production of ROS [21]. Moreover, liver injury induced by partial hepatectomy lead to induction of S100a8/a9 expression in hepatocytes, suggesting a role in liver regeneration [22]. Hitherto, we do not know whether the impact on pathological conditions of liver injury and carcinogenesis is mediated by well-known functions of S100a8/a9 on the amplification of pro-inflammatory signals, including immune cell recruitment and activation [15,23], because informative in vivo studies are still missing.
In contrast to other in vivo studies focusing on the function of heavily elevated systemic S100a8/a9 levels in settings of acute or chronic inflammation [18,24], we have established a conditional mouse model under the control of doxycycline to specifically investigate the effects of local S100a8/a9 overexpression without inducing a pathologic situation. In the present study we show that the simultaneous expression of S100a8 and S100a9 transgenes in hepatocytes leads to a constant homeostatic mobilization of neutrophils from the bone marrow via local induction of Cxcl1 expression. Importantly, the enrichment of the neutrophils occurs without activation and therefore hints to a so far unknown mechanism by which epithelial-derived S100a8/a9 may increase the surveillance capacity of an organism by innate immune cells.
Conditional and hepatocyte-specific transgene expression in TgS100a8a9hep mice
We established a new mouse line (TgS100a8a9hep) with conditional and hepatocyte-specific co-expression of S100a8 and S100a9 transgenes by using the Tetracyclin-inducible expression system (Tet-Off sytem) [25] to investigate the role of epithelial-derived S100a8/a9 under conditions of tissue homeostasis as well as pathological conditions. TgS100a8a9hep mice were generated by breeding of the hepatocyte-specific transactivator (tTA) mouse line TALap1 with the TetOa8a9 transgenic mouse line, containing the Myc/His-tagged S100a8 (tgS100a8) and S100a9 (tgS100a9) transgenes under the control of a tTA-dependent and bidirectional promoter (Figure 1A). First, tissue-specific expression of tgS100a8 and tgS100a9 transcripts was confirmed by semi-quantitative PCR with cDNA from different tissues derived of TgS100a8a9hep mice. Strong expression of both transgenes was detected in the liver, while minor tgS100a9 transcript levels were also found in brain and adipose tissue, in line with published data on the TALap1 transgene expression pattern (Figure 1B) [25]. In contrast, back skin, lung, heart, thymus, spleen, kidney, stomach, lymph nodes, and bone marrow displayed no expression for both transgenes (Figure 1B, Additional file 1: Figure S1A, data not shown). Quantification of tgS100a8 and tgS100a9 transcripts in livers from six month-old TgS100a8a9hep mice by qRT-PCR revealed a similar expression level compared to younger animals, demonstrating a persistent transgene expression over time (Additional file 1: Figure S1B). To confirm conditional transgene expression, TgS100a8a9hep mice were treated with or without doxycycline for three weeks, and abolished transgene expression upon doxycycline treatment was measured by qRT-PCR as compared to control-treated mice (Figure 1C). Finally, expression of tgS100a8 and tgS100a9 proteins was found in TgS100a8a9hep but not in Control mice by Western blot analysis, following enrichment from whole liver lysate (Figure 1D). However, immunohistochemical (IHC) staining of liver tissue sections did not show a strong signal for tgS100a8 and tgS100a9 proteins in hepatocytes of TgS100a8a9hep mice, whereas S100a8 and S100a9 positive immune cells were easily detectable (data not shown), suggesting that only minor amounts of transgenic proteins were produced. In line with this data, an S100a8/a9 heterodimer-specific ELISA revealed comparable amounts of the S100a8/a9 protein complex in serum samples of peripheral blood from Control and TgS100a8a9hep mice (Figure 1E). In summary, TgS100a8a9hep mice were characterized by a conditional and tissue-specific transgene expression, which resulted in a low but persistent tgS100a8 and tgS100a9 protein level in the liver under conditions of normal tissue homeostasis.
Additional file 1. Figure S1.TgS100a8 and tgS100a9 expression in TgS100a8a9hepmice. Expression of tgS100a8 and tgS100a9 were analysed by qRT-PCR with (A) cDNA of different tissues (liver, lymph nodes, and bone marrow) from 8 weeks old TgS100a8a9hep mice (n=3) or with (B) liver cDNA from two (n=5) and six (n=3) month old TgS100a8a9hep mice; values were normalized to age-matched Control animals; mean, +SD.
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Figure 1. Hepatocyte-specific and conditional S100a8 and S100a9 transgene expression in TgS100a8a9hepmice. (A) Schematic representation of the transgene construct: the transcriptional transactivator (tTA) is expressed under the control of the hepatocyte-specific promoter Lap1 (TALap1), binds to and activates the tetracycline operator (TetO), which drives the simultaneous expression of S100a8-myc/his and S100a9-myc/his transgenes (TetOa8a9). Doxycycline interferes with the binding of tTA to the TetO and inhibits transgene expression. (B) Tissue-specific tgS100a8 and tgS100a9 expression was determined by semi-quantitative PCR in different tissues from TgS100a8a9hep mice using specific primers for transgenic S100a8 and S100a9 transcripts. Hprt1 levels served as control for cDNA quantity and quality. (C) TgS100a8a9hep mice were treated with (+dox) or without (-dox) doxycycline (10 μg/ml) for three weeks and transgene expression was assessed by qRT-PCR using liver cDNA. The mean value +SD of n=3 animals per group is depicted. (D) Whole liver lysates from a TgS100a8a9hep mouse (dTg) and a single TetOa8a9 transgenic (sTg) control were purified with an anti-Myc-tag IP Kit and expression of transgenic proteins was monitored by Western blot analysis using a His-tag specific antibody. HeLa cells were transiently transfected with S100a8 and S100a9 expression vectors and whole cell lysate was used as a positive control (Co). (E) S100a8/a9 protein was quantified by a heterodimer-specific ELISA in serum of Control (n=7) and TgS100a8a9hep mice (n=6); mean +SD.
TgS100a8a9hep mice develop a systemic enrichment in neutrophils
The S100a8/a9 protein complex is associated with inflammation [7] especially in the context of leukocyte chemoattraction and activation [14,26]. To investigate the impact of transgene expression on normal liver homeostasis, we analyzed tissue sections from eight week-old Control and TgS100a8a9hep mice. Staining with hematoxylin and eosin (HE) revealed no obvious difference in liver architecture between TgS100a8a9hep and Control littermates (Figure 2A). Lack of liver damage in response to tgS100a8 and tgS100a9 expression was further confirmed by similar AST and ALT levels in serum samples from Control and TgS100a8a9hep mice (Additional file 2: Figure S2A, B). In addition, immunohistochemical staining with anti-HNE antibodies revealed no sign of increased ROS levels in TgS100a8a9hep as compared to Control livers (Additional file 2: Figure S2C). Moreover, IHC staining for the monocyte-specific antigen F4/80 revealed a comparable number of Kupffer cells in both genotypes (Figure 2A). In contrast, the staining for Gr1, a marker specific for granulocytes as well as monocytes and myeloid derived suppressor cells (MDSCs), displayed a significant increase in Gr1-positive immune cells in TgS100a8a9hep mice as compared to Control mice (Figure 2A and B).
Additional file 2. Figure S2. Analysis of additional parameters of inflammation and liver damage in TgS100a8a9hepmice. (A) ALT and (B) AST serum levels in 8 weeks old Control and TgS100a8a9hep mice (n=4). Mdr2-/- mice served as positive control; mean, +SD. (C) Liver sections from Control and TgS100a8a9hep mice were stained by IHC with specific antibodies for HNE adducts. Liver sections from Mdr2-/- mice served as positive control for the staining. Representative images from at least n=3 mice are shown with red staining for HNE and counterstaining with hematoxylin. Bar represent 200 μm. (D) Relative levels of endogenous S100a8 and S100a9 transcripts was measured by qRT-PCR with liver cDNA from Control (n=9) and TgS100a8a9hep (n=10) mice; mean, +SD, students t-test, *p≤0.05.
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Figure 2. Histological characterization of TgS100a8a9hepliver sections. (A) Tissue sections from Control and TgS100a8a9hep mice were stained with hematoxylin and eosin (HE) or by IHC with specific antibodies for monocytes (F4/80) and granulocytes (Gr1). Representative images from at least n=5 mice are shown with red staining for F4/80 or Gr1, respectively, and counterstaining with hematoxylin. Bars represent 200 μm. Gr1-positive (B) and S100a9-positive (C) immune cells from five randomly taken pictures (magnification 50x) from Control (n=9) and TgS100a8a9hep (n=10) were quantified. Box-plot shows median, 25 % and 75 % quartile (light grey box), 5 % and 95 % percentile (bars), students t-test, ***p≤0.001.
IHC staining of liver sections with specific antibodies for S100a8 and S100a9, respectively, well-characterized markers for granulocytes and circulating monocytes [3,27], and subsequent quantification of S100a8 and S100a9 positive immune cells confirmed a significant increase of this subpopulation of immune cells (Figure 2C, data not shown). Indeed, comparison of the relative amount of Gr1- and S100a9-positive cells in the liver of Control and TgS100a8a9hep mice revealed a high correlation coefficient (R2=0.95). Moreover, qRT-PCR analysis with cDNA of liver samples and primers recognizing selectively endogenous but not transgenic transcripts confirmed increased S100a8 and S100a9 expression in TgS100a8a9hep compared to Control mice (Additional file 2: Figure S2D). In summary, our data supported a causal link between transgene expression and the accumulation of a distinct Gr1-positive immune cell subpopulation of the monocyte lineage in the liver of TgS100a8a9hep mice.
To investigate whether the accumulation of this immune cell subpopulation increased with age or caused an alteration in liver architecture, liver sections of six month-old Control and TgS100a8a9hep mice were stained with HE and for Gr1-positive cells by IHC staining. A substantial increase in Gr1-positive cells was still detectable in liver sections of six month-old TgS100a8a9hep as compared to Control mice and no obvious difference was found when compared to younger TgS100a8a9hep animals (Additional file 3: Figure S3).
Additional file 3. Figure S3. Histological and immunohistochemical characterization of six month old TgS100a8a9hepmice. Liver sections from six month old Control and TgS100a8a9hep mice were stained with hematoxylin and eosin (HE) or by IHC with a specific antibody for granulocytes (Gr1). Representative images are shown with red staining for Gr1 (n=2 mice per group), and counterstaining with hematoxylin. Bars represent 200 μm.
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It is worth mentioning that liver sections of TgS100a8a9hep mice exhibited an equal distribution of Gr1-positive cells, suggesting that these cells were not actively recruited to the liver parenchyma but rather accumulated in the liver sinusoids due to increased amounts in the circulating blood. Accordingly, different tissues from Control and TgS100a8a9hep mice were analyzed for the expression of endogenous S100a8 and S100a9 transcripts by qRT-PCR, which was used as surrogate marker for granulocytes. Elevated expression of endogenous S100a8 transcript was detected in liver, brain, kidney, and heart from TgS100a8a9hep (n=4) as compared to Control mice (n=3), whereas the expression of S100a8 was not altered in spleen, thymus, stomach, and adipose tissue (Figure 3A). Similar results were measured for endogenous S100a9 transcript levels by qRT-PCR (Additional file 4: Figure S4A), indicating a specific increase in the amount of S100a8/a9 positive immune cells in highly perfused tissues. In line with this assumption, an almost 2.5-fold enrichment of neutrophils, but not eosinophils, basophils, monocytes, or lymphocytes was found in peripheral blood counts of TgS100a8a9hep as compared to Control mice (n=4, Figure 3B, Additional file 4: Figure S4B). Elevated levels of neutrophils were further confirmed by flow cytometric analysis using an antibody for Ly-6G, a specific marker for neutrophils [28,29], in combination with antibodies for Cd11b and Gr1 to distinguish the neutrophil subpopulation (Cd11b+/Gr1+/Ly-6G+) from other cell types (Cd11b+/Gr1+/Ly-6G-) including monocytes and monocytic MDSCs [28,30]. The total percentage of Gr1+/Ly-6G+ neutrophils within the Cd11b+ population was significantly increased in TgS100a8a9hep as compared to Control mice (n=7, Figure 3C), whereas the relative number of the Gr1+/Ly-6G- subpopulation was not affected by the transgene expression (Figure 3D). To exclude the possibility that Gr1-specific antibodies interfered with binding of anti-Ly-6G to its dedicated epitope, staining for neutrophils using an antibody specific for Ly-6C instead of anti-Gr1 was performed on wild type blood cells. Comparable amount of neutrophils were found with both staining procedures (Additional file 4: Figure S4C-E). In summary, our data demonstrate an age-independent systemic increase of neutrophils in the circulating blood of TgS100a8a9hep mice, which resulted in the accumulation of these cells in peripheral tissues with a strong blood supply, but without obvious features of inflammation or signs of pathological condition.
Additional file 4. Figure S4. Systemic enrichment of neutrophils in TgS100a8a9hepmice. (A) Relative expression of endogenous S100a9 transcripts was determined by qRT-PCR using cDNA from different tissues of Control (n=3) and TgS100a8a9hep (n=4) mice. Mean +SD, students t-test, *p≤0.05, **p≤0.01, n.s. not significant. (B) Absolute number of neutrophils in peripheral blood from Control and TgS100a8a9hep mice (n=4) as measured by means of blood counts. (C-E) Peripheral blood from Control mice (n=8) was subjected to tri-color staining using antibodies against Cd11b (APC), Ly-6G (FITC), and Gr1 (PE) or Ly-6C (PE), respectively. Representative images of either (C) Gr1/Ly-6G or (D) Ly-6C/Ly-6G populations are shown. (E) Graph shows the total percentage of Gr1+/Ly-6G+ cells and Ly-6C+/Ly-6G+ cells from all Cd11b+ cells in peripheral blood; +SD.
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Figure 3. Increased amount of neutrophils in peripheral tissue and blood samples. (A) Relative expression of endogenous S100a8 transcripts was determined by qRT-PCR using cDNA from different tissues of Control (n=3) and TgS100a8a9hep (n=4) mice. Mean +SD, students t-test, *p≤0.05, **p≤0.01, n.s. not significant. (B) Leukocyte subpopulations in the peripheral blood from Control and TgS100a8a9hep mice (n=4) were quantified and depicted as relative cell number. Box-plot shows median, 25 % and 75 % quartile (light and dark grey box), students t-test, **p≤0.01. (C-D) Peripheral blood from Control and TgS100a8a9hep mice (n=7) was subjected to tri-color staining using antibodies against Cd11b (APC), Gr1 (PE), and Ly-6G (FITC). Graphs show the total percentage of Gr1+/Ly-6G+ cells (C) and Gr1+/Ly-6G- cells (D) from all Cd11b+ cells in peripheral blood. Box-plot shows median, 25 % and 75 % quartile (light grey box), students t-test, *p≤0.05, n.s. not significant.
Systemically enriched neutrophils in TgS100a8a9hep mice are not activated
Since TgS100a8a9hep mice did not develop an obvious pathological phenotype with age, we investigated the activation state of the systemically enriched neutrophil subpopulation. Upregulation of Mac-1 (Cd11b/Cd18) and ectodomain shedding of L-Selectin (Cd62L), two frequently used activation markers for neutrophils [31], were determined by flow cytometry on peripheral blood neutrophils (Gr1+/Ly6G+ for Cd11b; Cd11b+/Ly6G+ for L-Selectin) derived from Control and TgS100a8a9hep animals (n≥5). Importantly, no difference in the mean intensity was detectable for Cd11b and L-Selectin surface expression, while Cd11b upregulation and L-Selectin shedding was easily detectable upon TPA-stimulation of isolated neutrophils (Figure 4A, B and Additional file 5: Figure S5), suggesting an accelerated mobilization but no further activation of neutrophils in TgS100a8a9hep mice.
Additional file 5. Figure S5. Molecular markers of neutrophil activation in TgS100a8a9hepmice. Blood neutrophils (Gr1+/Ly6G+ or Cd11b+/Ly6G+) from Control and TgS100a8a9hep mice were stained with specific antibodies for Cd11b (APC) and L-Selectin (PE), or the respective isotype control antibody. Representative histograms of Cd11b (A) and L-Selectin (B) stainings are shown for Control (red) and TgS100a8a9hep (green) mice, including the staining with their respective isotype control (black).
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Figure 4. Molecular markers of neutrophil activation in TgS100a8a9hepmice. Blood neutrophils (Gr1+/Ly-6G+ or Cd11b+/Gr1+) from Control and TgS100a8a9hep mice or isolated neutrophils treated with 10 μg/ml TPA for 5 min (Pos) were stained with specific antibodies for (A) Cd11b (APC) and (B) L-Selectin (FITC), or the respective isotype control antibody. The mean fluorescence intensities from Control (Cd11b n=9; L-Selectin n=5), TgS100a8a9hep (Cd11b n=8; L-Selectin n=5) and Pos (n=3) samples were determined and depicted as means +SEM.
Altered expression of the chemokine Cxcl1 in the liver of TgS100a8a9hep mice
As neutrophils have a relatively short life span in peripheral blood, continuous renewal by mobilization from the bone marrow is mandatory [32]. This homeostatic release from the bone marrow is tightly regulated by an equilibrium of retention and mobilization factors either keeping the neutrophils in the hematopoietic cords of the bone marrow or mediating the transition across the sinusoidal endothelium into the circulating blood [32]. Recent studies demonstrated the importance of several chemokines such as Cxcl1, Cxcl2, and Csf3 (G-Csf) and the interplay with their cognate receptors Cxcr2 and Cxcr4 on the surface of neutrophils in the regulation of homeostatic neutrophil mobilization [33,34]. Moreover, the factors Csf1 (M-Csf) and Csf2 (Gm-Csf) have been related to neutrophil mobilization under homeostasis as well [32]. Although, several reports showed S100a8/a9-mediated chemoattraction of neutrophils [14,16,24,26], a direct involvement of transgenic proteins in neutrophil mobilization in TgS100a8a9hep mice was unlikely as no systemic difference in S100a8/a9 protein serum levels was detected (Figure 1E). Consequently, we hypothesized that local expression of transgenic S100a8 and S100a9 in the liver of TgS100a8a9hep mice induced the expression of one or more factors, which in turn were able to mediate neutrophil mobilization from the bone marrow. First, we analyzed cDNA from livers of Control and TgS100a8a9hep mice by qRT-PCR for the expression of Csf1, Csf2, Csf3, Cxcl1, and Cxcl2. Transcripts of Csf2 and Csf3 were not detectable (data not shown), whereas the expression of Cxcl2 and Csf1 revealed no significant difference between TgS100a8a9hep and Control livers (Figure 5A). Interestingly, Cxcl1 transcripts were significantly upregulated in TgS100a8a9hep as compared to Control mice (Figure 5A).
Figure 5. Chemokine levels in liver, primary hepatocytes, and serum of Control andTgS100a8a9hepmice. (A) Relative expression of Cxcl1, Cxcl2 and Csf1 was determined by qRT-PCR analysis on liver cDNA from Control (n=6) and TgS100a8a9hep (n=9) mice and is depicted as mean +SD. (B) Primary hepatocytes were isolated from Control and TgS100a8a9hep mice and cultivated in vitro for 24 hours. Relative expression of Cxcl1, Cxcl2, Csf1 was measured by qRT-PCR on cDNA from primary hepatocytes. Two biological replicates for each group were measured in triplicates and means are depicted +SD, students t-test, *p≤0.05. (C) Protein levels of Cxcl1, Cxcl2, and Csf3 in serum samples from Control and TgS100a8a9hep (n≥4) mice were measured by ELISA. Mean +SD, students t-test, *p≤0.05.
To address the question whether hepatocytes are the source of increased Cxcl1 transcript levels in TgS100a8a9hep mice, livers of Control and TgS100a8a9hep mice (n=3 each) were perfused with collagenase and the cell suspension was cultivated in vitro for 24 hours to obtain primary hepatocytes. One aliquot of the cell suspension was directly taken after enzymatic digestion and a significant increase in Cxcl1 transcript levels in the liver of TgS100a8a9hep mice was confirmed by qRT-PCR (Additional file 6: Figure S6A). Subsequent qRT-PCR analysis with cDNA from primary hepatocytes, which were cultured in vitro for 24 hours revealed a strong expression in S100a8 and S100a9 transgenes in TgS100a8a9hep hepatocytes and a significant increase in the expression of Cxcl1 in TgS100a8a9hep as compared to Control hepatocytes, whereas Cxcl2 and Csf1 were not affected (Figure 5B and Additional file 6: Figure S6B). These data strongly suggest that hepatocytes are the main source of increased Cxcl1 transcription in the liver of TgS100a8a9hep mice.
Additional file 6. Figure S6.Cxcl1 and tgS100a8/a9 expression in primary hepatocytes from Control and TgS100a8a9hepmice. (A) Relative Cxcl1 transcript levels was determined by qRT-PCR analysis with cDNA from cell suspension derived from collagenase perfused livers of Control and TgS100a8a9hep mice. (B) Primary hepatocytes were cultivated in vitro for 24 hours and relative levels of tgS100a8 and tgS100a9 transcripts were measured by qRT-PCR. Two biological replicates for each group were measured in triplicates and means are depicted +SD, students t-test, **p≤0.01.
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Finally, we analyzed serum and liver samples of Control and TgS100a8a9hep mice by enzyme-linked absorbent immunoassays (ELISA) for Cxcl1, Cxcl2, and Csf3 protein levels. Substantial levels of Cxcl1 and Csf3 were measured in both groups, whereas only low amounts were detected for Cxcl2 (Figure 5C). We found no major difference in Cxcl2 and Csf3 protein levels, but in accordance with the qRT-PCR data, Cxcl1 protein levels were significantly increased in TgS100a8a9hep as compared to Control mice (Figure 5C and Additional file 7: Figure S7).
Additional file 7. Figure S7. Cxcl1 protein expression in liver lysates from Control and TgS100a8a9hepmice. Protein levels of Cxcl1 in liver lysate from Control and TgS100a8a9hep mice (n=4) were measured by ELISA. Mean +SD, students t-test, **p≤0.01.
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Enhanced Cxcl1 expression and neutrophil enrichment in TgS100a8a9hep mice depends on transgene expression
Control (Co) and TgS100a8a9hep (Tg) animals were treated either with (Tg +dox, Co +dox) or without (Tg -dox, Co -dox) doxycycline-containing drinking water for three weeks to investigate, whether the observed increase in Cxcl1 levels and systemic enrichment of neutrophils critically depends on the presence of hepatocyte-specific tgS100a8 and tgS100a9 expression in adult mice. qRT-PCR analysis on livers from four animals per group, respectively, confirmed a significant increase in Cxcl1 transcript levels in Tg -dox mice as compared to Co -dox animals. While doxycycline treatment had no impact on Cxcl1 expression in Co +dox mice, the Cxcl1 transcript levels were reduced to basal levels in Tg +dox mice upon doxycycline treatment (Figure 6A). In line with reduced Cxcl1 expression, we found a significant decreased in the amount of peripheral neutrophils in the blood of Tg +dox mice (Figure 6B, Additional file 8: Figure S8), as measured by FACS analysis and blood counts, accompanied by reduced neutrophil numbers in liver sections of Tg +dox as compared to Tg -dox littermates (Figure 6C). In contrast, doxycycline treatment did neither affect neutrophil numbers nor the number of Cd11b+/Gr1+/Ly6G- cells in Co +dox mice (Figure 6, data not shown). Absolute numbers of neutrophils in blood counts from Control and TgS100a8a9hep mice with and without doxycycline treatment revealed similar results (Additional file 8: Figure S8).
Additional file 8. Figure S8. Peripheral neutrophils in Control and TgS100a8a9hepmice treated with doxycycline. Control (Co) and TgS100a8a9hep (Tg) mice were treated with (Co +dox, Tg +dox) or without (Co -dox, Tg -dox) doxycycline (10 μg/ml) containing drinking water. Peripheral blood neutrophils were measured by means of blood counts and depicted as absolute numbers. Box-plot shows median, 25 % and 75 % quartile (light grey box).
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Figure 6. Measurement of neutrophil numbers and Cxcl1 expression in Control and TgS100a8a9hepmice treated with doxycycline. Control (Co) and TgS100a8a9hep (Tg) mice were treated with (Co +dox, Tg +dox) or without (Co -dox, Tg -dox) doxycycline (10 μg/ml) containing drinking water. (A) Relative expression of Cxcl1 was determined by qRT-PCR on liver cDNA. Mean +SD, students t-test, *p≤0.05, **p≤0.01, n.s. not significant. (B) Peripheral blood cells were stained with specific antibodies for Cd11b (APC), Gr1 (PE) and Ly-6G (FITC) followed by flow cytometry analysis. Box-plot shows the relative fold increase of Gr1+/Ly-6G+ population from all Cd11b+ cells in peripheral blood; Median, 25% and 75% quartile (light grey boxes), students t-test, *p≤0.05. (C) IHC staining with Gr1-specific antibodies was performed on liver sections and representative images (n=4 animals per group) are shown with red staining for positively stained cells and counterstaining with hematoxilin. Bars represent 200 μm.
Taken together our data confirmed that the increase in Cxcl1 expression, which is accompanied by systemic enrichment of neutrophils in TgS100a8a9hep mice critically depends on conditional S100a8 and S100a9 transgene expression in the liver.
The p38 MAPK pathway is inhibited inTgS100a8a9hephepatocytes and represents a negative regulator of Cxcl1 expression
In order to unravel molecular mechanisms of transgene induce Cxcl1 expression, we prepared protein lysate from freshly isolated primary hepatocytes derived from Control and TgS100a8a9hep liver. Western Blot analysis revealed similar expression and phosphorylation of p65, c-Jun, Stat3, and ERK1/2, but a substantial decrease in phospho-p38 protein levels was evident in TgS100a8a9hep as compared to Control hepatocytes (Figure 7A). Densitometric quantification of phosphorylated and total p38 protein levels confirmed a 50% reduced phosphorylation due to transgene expression (data not shown). To further confirm the role of phosphorylated p38 as a negative regulator for Cxcl1 expression, freshly isolated wild type primary hepatocytes were treated with the p38 MAPK-specific inhibitor SB203580 for 5 hr and qRT-PCR analyses revealed a significant increase of Cxcl1 transcript levels in hepatocytes with impaired p38 MAPK activity as compared to DMSO treated cells (Figure 7B). In contrast, Cxcl2 and Csf1 transcript levels were significantly decreased upon p38 inhibition, which is in line with our previous observation that tgS100a8/a9 expression specifically induces Cxcl1 expression in hepatocytes from TgS100a8a9hep mice (Figure 5B). In summary, these data demonstrate that impaired p38 MAPK activity in the presence of tgS100a8/a9 expression triggers a specific induction of Cxcl1 expression in hepatocytes.
Figure 7. Involvement of signaling pathways in Cxcl1 upregulation in hepatocytes. (A) Primary hepatocytes were isolated from Control and TgS100a8a9hep mice (n=2). Expression of p65, c-Jun, Stat3, p38, ERK1/2, and the corresponding phosphorylated forms was monitored by Western Blot analysis. (B) Primary hepatocytes were treated either with SB203580 or with (DMSO) for 5 h and relative expression of Cxcl1, Cxcl2, and Csf1 was determined by qRT-PCR; n=5, Mean +SD, students t-test, **p≤0.01.
A broad range of experimental studies demonstrate a direct link between S100a8/a9 protein expression and acute and chronic inflammation as well as inflammation-induced carcinogenesis [8]. In fact, S100a8/a9 is involved in several steps of the inflammatory response including leukocyte differentiation and chemoattraction, transendothelial migration, activation of immune and epithelial cells, and the induction of pro-inflammatory mediators [7]. Thereby S100a8/a9 might promote an inflammatory feed-forward loop, which facilitates the transition to a chronic inflammation. An established chronic inflammation strongly increases the likelihood of pathologic conditions such as tumor formation and is even considered as a hallmark of carcinogenesis [35]. Indeed in vivo studies with S100a9-/- animals highlighted a crucial role for S100a8/a9 in the promotion of inflammation and inflammation-induced carcinogenesis in models of antigen-induced arthritis and colon carcinogenesis, respectively [18,19]. However these studies focused on the role of S100a8/a9 in the differentiation and function of myeloid-derived S100a8/a9. In contrast, no causal connection of epithelial-derived S100a8/a9 and the initiation and maintenance of a chronic inflammation and subsequent tumor-development have been demonstrated yet.
In order to investigate the role of epithelial-derived S100a8/a9 in liver homeostasis, we generated TgS100a8a9hep mice with conditional and hepatocyte-specific S100a8 and S100a9 expression. The initial characterization revealed persistent transgene expression in liver. Furthermore the conditional tgS100a8/a9 expression in TgS100a8a9hep mice was confirmed via doxycycline treatment. Additionally, transgenic protein was detected in purified whole liver lysates from TgS100a8a9hep mice, whereas the serum levels of total S100a8/a9 were unchanged in TgS100a8a9hep mice compared to Controls. In summary the TgS100a8a9hep mice showed conditional and hepatocyte-specific expression of tgS100a8/a9 transcripts, which was not sufficient to reach a systemic increase of total S100a8/a9. Interestingly, all animal models, which were used so far to show S100a8/a9-dependent immune cell recruitment and activation accompanied by pathologic conditions were characterized by a strong S100a8/a9 overexpression [14,16,23,24]. In contrast, the use of the TgS100a8a9hep mouse model enabled for the first time a detailed analysis of physiological and pathophysiological alterations in settings of low and locally restricted S100a8/a9 induction in vivo.
S100a8/a9 has been shown to act as a potent chemoattractant and activator for monocytes and neutrophils [14,21,23]. Therefore, tgS100a8/a9 expression might be expected to induce pathologic changes affecting liver homeostasis. However, TgS100a8a9hep mice showed neither obvious differences in liver architecture and liver damage nor changes in the number of resident Kupffer cells. Interestingly, TgS100a8a9hep animals developed a significant enrichment of Gr1-positive immune cells in the liver as compared to Control mice. Further characterization showed that this immune cell subpopulation was positive for endogenous S100a8 and S100a9 proteins and transcripts, and importantly, aged TgS100a8a9hep animals exhibited a comparable enrichment of this specific immune subpopulation, supporting the existence of a constitutive signal in TgS100a8a9hep mice, which leads to a sustained increase of these cells. Of note, the Gr1-positive subpopulation did not infiltrate into the liver parenchyma but rather accumulated in the blood and therefore appeared in the sinusoids of the liver. Therefore we hypothesized a systemic accumulation of Gr1-positive cells in TgS100a8a9hep mice. Indeed, qRT-PCR analysis revealed an elevation of endogenous S100a8/a9 transcripts, which was used as a surrogate marker for the affected subpopulation, also in other perfused tissues including brain, kidney, and heart. This further supported the hypothesis of a systemic enrichment in TgS100a8a9hep mice, which was further confirmed by blood counts and flow cytometric analysis on peripheral blood samples. Although spleen is a highly perfused organ, it serves as marginating pool for granulocytes, ensuring rapid mobilization of innate immune cells upon infection and inflammation to facilitate an immediate inflammatory response [36]. Consequently, the overall amount of S100a8/a9 is very high and the difference due to the enriched population in TgS100a8a9hep mice was not detectable.
Neutrophils circulating in the blood stream under healthy conditions, serve as first defense line against pathogens, and represent key players in sterile inflammatory diseases by secreting a plethora of different pro-inflammatory mediators [37]. Since the TgS100a8a9hep mice developed no obvious pathological phenotype over time it was questioned whether the neutrophils were fully activated. Indeed, flow cytometric analysis on neutrophils of TgS100a8a9hep mice revealed neither an increase in Cd11b expression nor ectodomain shedding of L-Selectin, which are well-known activation markers of neutrophils [31,38]. Together, these data convincingly show that the enriched neutrophils in the TgS100a8a9hep mice are not activated, and additional signals are missing to induce an acute and/or chronic inflammation.
Injection of high amounts of S100a8/a9 was shown to directly act as a chemoattractant for neutrophils and lead to tissue-specific recruitment but was also reported as an activator of neutrophils [14,23]. Furthermore, a causal role for S100a8 in inducing myeloid cell migration has been demonstrated without further activation [39]. However TgS100a8a9hep mice showed a systemic enrichment of neutrophils without activation and site-specific recruitment. This argues for a so far unknown mechanism of S100a8/a9, which might be only visible under conditions when tgS100a8/a9 expression is locally restricted to the liver without potent systemic accumulation. Hence we assumed that a mechanism, which is implicated in regulation of circulating neutrophils under homeostasis, was affected in TgS100a8a9hep mice resulting in elevated neutrophil levels. The number of circulating neutrophils is mainly regulated via a constant release from the bone marrow compartment. This homeostatic release is realized either by Csf3, which is able to interfere with the Cxcl12-Cxcr4 mediated neutrophil retention [33,34,38], or by Cxcl1 and Cxcl2, which directly act as chemoattractants for neutrophils via Cxcr2 [40]. Moreover, experimental evidence suggests that other factors e.g. Csf1 and Csf2 might be involved in neutrophil mobilization under homeostasis [40,41]. Interestingly, only Cxcl1 was increased in the liver and serum of TgS100a8a9hep compared to Control mice, whereas other factors were either not detectable or were not altered. Moreover, Cxcl1 was overexpressed in primary hepatocytes isolated from TgS100a8a9hep mice, supporting that an increase in Cxcl1 expression is at least in part mediated by hepatocytes.
In line with our findings, recent reports demonstrated that administration of recombinant Cxcl1 in mice results in a three- to four-fold increase in the number of circulating neutrophils due to release from the bone marrow [34,42]. Accordingly, transgenic mice overexpressing Cxcl1 in different tissues showed elevated numbers of circulating neutrophils [43]. It is worth mentioning that also Cxcl1-induced neutrophils showed no signs of further activation in terms of Cd11b upregulation and L-Selectin shedding [31]. On the other hand mice deficient for Cxcr2 displayed an abnormal retention of neutrophils in the bone marrow whereas the numbers of circulating neutrophils largely declined [33].
To finally prove the causal link between transgene expression and the Cxcl1 induction as well as the accumulation of neutrophils in TgS100a8a9hep mice, the transgene expression was repressed by doxycycline treatment. Indeed, TgS100a8a9hep mice with repressed transgene expression exhibited baseline Cxcl1 transcript levels in the liver, accompanied by a normal number of neutrophils in peripheral blood. These data supported the assumption that the expression of Cxcl1 transcripts is causally linked to tgS100a8/a9 expression in the liver and that the accelerated mobilization of neutrophils from bone marrow is mediated by the specific increased of Cxcl1 expression in the liver of TgS100a8a9hep mice (Figure 8).
Figure 8. Model of S100a8/a9-Cxcl1-mediated neutrophil enrichment in TgS100a8a9hepmice. The S100a8/a9 protein complex is produced in the liver of TgS100a8a9hep mice and induces a specific expression of the neutrophil chemoattractant Cxcl1 in hepatocytes, which in turn leads to an increased pool of Cxcl1 protein in the serum of transgenic mice. Consequently, Cxcl1 enhances the release of neutrophils from the bone marrow and increases the number of circulating non-activated neutrophils, which also appear in distant organs such as liver.
The expression of Cxcl1 is regulated by several transcription factors, including NF-κB [44], AP-1 [45], and Stat3 [46], which facilitate a rapid activation upon inflammatory stimulation. S100a8/a9 expressed by hepatocytes in TgS100a8a9hep mice may be secreted in minor amounts and act in an autocrine manner via binding to Rage or Tlr4, both well established membrane receptors for extracellular S100a8/a9 [19,47]. Engagement of these receptors has been shown to induce relevant transcription factors for induced Cxcl1 transcription as well as their upstream signaling pathways, such as p38 and ERK1/2 MAPKs [48-50]. Western Blot analysis of primary hepatocytes revealed a substantial decrease in activation of the p38 MAPK pathway whereas other pathways were not altered. Inhibition of p38 activity was shown previously by our group and others in situations of intracellular S100a8/a9 expression [21,51]. In contrast, extracellular S100a8/a9 rather lead to a positive regulation of p38 [17,51]. Together with the finding that S100a8/a9 serum levels are unchanged in TgS100a8a9hep as compared to Control and increased Cxcl1 levels were observed in isolated hepatocytes our results point to an intracellular rather than an extracellular mode of action, how S100a8/a9 mediated Cxcl1 induction. Finally, inhibition of p38 MAPK lead to increased expression of Cxcl1 in primary hepatocytes by an as yet unknown mechanism indicating a role of p38 as a negative regulator for Cxcl1 expression under conditions without inflammatory stimuli.
TgS100a8a9hep mice with conditional and hepatocyte-specific expression of S100a8 and S100a9 served as an useful tool to unravel a so far unknown function of calprotectin on the mobilization of neutrophils. Our new findings have a major impact on the current view, how calprotectin may influence acute and chronic inflammation under pathological conditions. Ichikawa et al. [19] demonstrated in a model of colitis-associated carcinogenesis that myeloid-derived S100a8/a9 induces chemokine expression by tumor cells, including Cxcl1, which was associated with an accumulation of Cd11b+/Gr1+ immune cells. However, TgS100a8a9hep mice revealed a neutrophil mobilization upon expression of S100a8/a9 in non-transformed epithelial cells (Figure 8), and elucidated a complete new aspect of the role played by calprotectin in epithelial cells: S100a8/a9 may support an acute inflammatory response by increasing steady state levels of circulating neutrophils and thereby help to restore tissue homeostasis. However, under settings of sustained tissue damage the constitutive mobilization of neutrophils by a S100a8/a9-Cxcl1-dependent mechanism could enhance the risk for the development and maintenance of chronic inflammation.
Animals and animal work
Animals were maintained in a specific pathogen-free environment. All animal experiments were performed with age-matched male mice. The procedures for performing animal experiments were in accordance with the principles and guidelines of the Arbeitsgemeinschaft der Tierschutzbeauftragten in Baden-Württemberg and were approved by the German Regional Council in Karlsruhe.
TALap1 mice were described previously [25]. In order to generate the TetOa8a9 mouse line, a bidirectional Tet-inducible expression plasmid was cloned from the parental pBi-L (Clontech Laboratories) encoding S100a8-myc/his and S100a9-myc/his fusion constructs driven by Tet-responsive promoter. The linearized pBi-S100a8a9 construct was injected into blastocytes of C57B1/6 mice. Genomic integration was determined by Southern Blot analysis.
For the analysis TALap1 and TetOa8a9 single transgenic mice served as Control animals. For doxycycline treatment, 6 to 8 week-old animals received 10 μg/ml doxycycline 5% sucrose containing drinking water ad libitum for a period of 3 weeks. Primary mouse hepatocytes were isolated and cultured as described [52]. Briefly, 8 week-old mice were anesthetized by intraperitoneal injection of 5 mg/100 mg body weight 10% ketamine hydrochloride and 1 mg/100 mg body weight 2% xylazine hydrochloride. After opening the abdominal cavity, the liver was perfused at 37°C with EGTA buffer (0.6% Glucose (w/v), 105 mM NaCl, 2.4 mM KCl, 1.2 mM KH2PO4, 26 mM Hepes, 490 μM L-Glutamine, 512 μM EGTA, 15% (v/v) amino acid solution, pH 8.3) via the portal vein for 5 min and subsequently for 5 min with Collagenase buffer (0.6% Glucose (w/v), 105 mM NaCl, 2.3 mM KCl, 1.2 mM KH2PO4, 25 mM Hepes, 490 μM L-Glutamine, 5.3 mM CaCl2, 12% (v/v) amino acid solution, 365 μg/ml collagenase type I-A, pH 8.3) until disintegration of the liver structure was observed. The liver capsule was removed, and the cell suspension was filtered through a 100 μm mesh. Cells were washed and, subsequently, viability of cells was determined by trypan blue staining. Two million living cells were seeded on collagen type I-coated 6 cm dishes and harvested after 24 hr. For in vitro inhibition of p38 MAPK, freshly isolated primary hepatocytes were treated with SB203580 (10 μM) or its solvent (DMSO) and harvested after 5 hr.
Protein isolation and Western blot analysis
Preparation of whole cell extracts was done with radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% (v/v) sodium deoxylcholate, 1% (v/v) NP-40, proteinase inhibitor cocktail), and purification of Myc-tagged transgenic S100a8/a9 was performed with mammalian cMyc-tag/Co-IP Kit (Pierce) according to the manufacturer's instructions. Proteins were separated on a 15% SDS-polyacrylamide gel in SDS-sample buffer, transferred to nitrocellulose membrane (Optitran BA-S83) and incubated with specific primary and HRP-labeled secondary antibodies (DakoCytomation). The blots were developed using the Classic E.O.S. developer (Agfa). Primary antibodies are listed in Additional file 9 (Table S1).
Additional file 9. Table S1. Antibodies used for IHC, Western blotting, and flow cytometry.
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ELISAs for Cxc11, Cxcl2, G-Csf (all from R&D Systems), and S100A8/A9 (Immundiagnostik AG) were performed according to the manufacturer's instructions.
Immunohistochemistry and tissue staining
IHC analysis on paraffin sections was performed according to the manufacturer's instructions (Vector Laboratories), and sections were counterstained with hematoxylin. Primary and secondary antibodies are listed in Additional file 9 (Table S1). Stained tissue sections were visualized and photographed using a Leica DMLB microscope and Olympus XC50 camera at a magnitude of 100x. Representative pictures for each genotype are shown.
RNA preparation and quantitative real time polymerase chain reaction
Total RNA extraction was performed according to the manufacturer`s instructions using peqGOLD RNAPure (PeqLab Biotechnology). RNA was reverse transcribed using RevertAid H Minus-MuLV RT (Fermentas) and oligo-dT primers, followed by standard qRT-PCR performed on a detection system (StepOnePlus™; Applied Biosystems) using the Power SYBR® Green PCR Master Mix (Applied Biosystems). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed as described previously [53]. Primers used for qRT-PCR analysis are listed in Additional file 10 (Table S2). Target gene cycle of threshold values were normalized to the corresponding cycle of threshold values of Hprt using the change in cycle of threshold method.
Additional file 10. Table S2. Primer pairs used for qRT-PCR analysis.
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Preparation of blood samples and flow cytometry
For detection of immune subpopulations in peripheral blood, whole blood samples were mixed with EDTA (final 5 mM), subjected to erythrocyte lysis (ACK buffer, Lonza) and subsequently blockade of Fc-receptors (anti-Cd16/32, eBioscience). Staining was performed at 4°C for 30 min and cells were determined by flow cytometry analysis in FACSCalibur (BD Bioscience) using the software FlowJo (Tree Star, Inc.). Antibodies are listed in Additional file 9 (Table S1). As positive control for Cd11b upregulation and L-Selectin shedding, peripheral blood cells were treated with 10 μg/ml TPA for 5 min and stained as described.
Digital images and statistical analysis
Digital images were elaborated using Photoshop CS3 (Adobe); the luminosity of brightest and dimmest pixel in each channel were adjusted to obtain the best visual reproduction, taking care to maintain linearity in the brightness scale. Error bars represent SD except were indicated. Statistical evaluation of obtained data was performed with SigmaPlot 11.0 (Systat Software Inc.).
ALT: Alanine transaminase; APC: Allophycocyanin; AST: Aspartate transaminase; CD: Cluster of differentiation; Csf: Colony-stimulatory factor; Cxcl: C-X-C motif ligand; Cxcr: C-X-C motif receptor; ELISA: Enzyme-linked absorbent immunoassay; FITC: Fluorescein isothiocyanate; G-Csf: Granulocyte colony-stimulatory factor; Gm-Csf: Granulocyte macrophage colony-stimulating factor; HE: Hematoxylin and eosin; Hprt: Hypoxanthine-guanine phosphoribosyltransferase; ICAM: Intercellular adhesion molecule; IHC: Immunohistochemistry; IL: Interleukin; Lap1: Liver activator protein 1; Ly6: Lymphocyte antigen 6; Mac-1: Macrophage-1 antigen; M-Csf: Macrophage colony-stimulating factor; Mdr2: Multidrug resistance 2; MDSCs: Myeloid derived suppressor cells; NADPH: Nicotinamid adenosine dinucleotide phosphate; PE: Phycoerythrin; qRT-PCR: Quantitative real-time PCR; ROS: Reactive oxygen species; tetO: Tetracyclin-operator; TgS100a8a9hep: Hepatocyte-specific S100a8/a9 transgenic mouse; TNF: Tumor necrosis factor; tTA: Tetracyclin transactivator.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LW, JN, TP, CB, AD, SMan, and SMar performed experiments; LW analyzed results, made figures, and wrote the paper; LW, AV, UK, JH, and PA designed the research. All authors read and approved the final version of this manuscript.
We thank Angelika Krischke for excellent technical support and Julia Leibold for helpful discussion. This work was supported by the Cooperation in Cancer Research of the German Cancer Research Center and Israeli Ministry of Science, Culture and Sport (Ca-130 to PA, and JH, and Ca-147 to PA), the German Research Foundation (SFB Transregio 77, to SMar, AV, PA, and JH), the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Systems Biology (to PA and JH), the Federal Ministry of Science, Education and Art (MWK Excellence Cluster Initiative to PA and JH), the Foundation Tumorforschung Kopf-Hals e.V. (to JH), and the 'Virtual Liver' funding initiative of the German Federal Ministry of Education and Research (BMBF) (to SMan and UK).
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Am J Physiol Gastrointest Liver Physiol 2010, 299:G867-876. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
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text:
Seanan McGuire
Discount Armageddon (InCryptid #1)
Urban fantasy
March 6, 2012
Blurb via Goodreads:
Ghoulies. Ghosties. Long-legged beasties. Things that go bump in the night… The Price family has spent generations studying the monsters of the world, working to protect them from humanity-and humanity from them. Enter Verity Price. Despite being trained from birth as a cryptozoologist, she'd rather dance a tango than tangle with a demon, and is spending a year in Manhattan while she pursues her career in professional ballroom dance. Sounds pretty simple, right? It would be, if it weren't for the talking mice, the telepathic mathematicians, the asbestos supermodels, and the trained monster-hunter sent by the Price family's old enemies, the Covenant of St. George. When a Price girl meets a Covenant boy, high stakes, high heels, and a lot of collateral damage are almost guaranteed. To complicate matters further, local cryptids are disappearing, strange lizard-men are appearing in the sewers, and someone's spreading rumors about a dragon sleeping underneath the city…
I received the ARC for this one not long before Christmas 2011 – hello, awesome present! I finished it up right around New Year's. People. People, listen. Look, this is a really good book. I'm just going to say that I just about loved it to pieces, like the kinds of pieces one gets when they hug something breakable with super human strength and, oh, oops, there I went and broke that awesome thing. Guess it's a good thing this wasn't on the Kindle, then. Paperbacks are actually pretty forgiving when it comes to ridiculously overzealous hugs.
A quick, minor note on the cover – I remember hearing a few grumbles about the cover a while back, namely because of the heroine's dress, pose, etc. It's actually an accurate depiction. A reason for a plaid pink skirt, stockings – those shoes? The weapons? And her minimalistic top? Oh yes. It's a beautiful thing, trust me.
There's a few things I realized I almost desperately needed and/or loved after reading this book:
Sliding my legs out the window, I pushed off from the windowsill and fell into the dark.
1. Verity Price's unabashed love of leaping over tall buildings with little to no effort. Sure, she worked hard at building up the momentum and speed and courage (not to mention complete loony bin brains) necessary to do this while training with her siblings, but it's practically a major genetic trait that roars through her family. OK, so not an actual genetic trait, that was just a slight exaggeration. Still – I could not stop my grins of delight every time the woman just casually steps off a damn building or takes a wall with some flimsy fire escape at a dead run and ends up on top of New York City.
I myself would develop the two biggest left feet in the history of left feet and merely plummet to my death. Would make for a boring ass book. To say the least DAMMIT CHILD. (Sorry, kidlet got out of bed for millionth time, I corralled her back to bed – good times. DAMMIT CHILD might appear in more reviews in the future.)
Life as the chosen religious figure for a colony of cryptid mice can be a lot of things, but it's definitely never boring.
2. Aeslin mice. I'm a little afraid of explaining these too much; you should really experience them as much as possible for yourself. Suffice to say they are rodents, they are mighty and they shout, "HAIL!" upon seeing Verity. A lot. They are some of the best comedic relief I've ever had the pleasure of chortling at. Just picture it, three-inch-tall pillars of never ending enthusiasm – FOR YOU. They reminded me instantly of those furry mini aliens of Locker C18 in Men in Black II, which in my opinion is always a good thing.
"Remember, Very," Dad used to say when I whined about the goggles, "if your opponent has night vision and you've never bothered to learn the local landmarks by anything but sight, you're going to be in a bit of a pickle when it's time to avoid getting disemboweled."
3. Verity's family. They aided in making Verity what she is, which is one of the most unique and funny and kick-butt heroines I've ever had the pleasure of reading. Even though we don't see a whole whole lotta her fam, they are irresistibly interesting people. And it just so happens you can get a short story about some of them for free from the author.
"I assure you, ma'am, your capture was not my intention."
–Dominic De Luca upon setting Verity Price free of a snare.
4. The romance. It's not overbearing. It doesn't turn Verity into a poor, pitiful woman entirely dependent on the hero, Dominic De Luca. It also doesn't mean she can't take help when she needs it, but in terms of romance, it knows its place in this one and that is to enhance the rest of the book, not overtake it.
On another note, Dominic is an interesting lead guy and I was pleased at how well they immediately have this insurmountable conflict between them. Family feud-type dilemmas are one of my faves.
My name is Verity Price. I'm a cryptozoologist. And this is why I can never get a goddamn date on a Saturday night.
5. The worldbuilding. I already knew McGuire was wonderful at creating an entirely new and seriously creepy and eye-widening-exciting fae world in her October Daye series, but the way she reintroduces mythlogical and assorted popular creatures in this series is once again something to cheer for. And not only will you understand exactly what each one she encounters in this book is like, but you'll do so thanks to an incredible job at weaving it into the book's plot. There is some mild use of informational paragraphs, but not to the point that I felt bogged down or bored. It's yet another veeewwwwy intwesting urban fantasy world McGuire's given readers to play in.
"Oh, fuck it," I said, and strode into the blackness.
6. The book ends. While we know this is a series, and you'll definitely see threads that start a series arc, this book has an ending, not a Ha ha, suckers, you've got another year to wait and see what happened to the main characters with that thingy thang on the last page. No readers have been taunted at the ending of this book, I can guarantee it.
OK, so that, basically, is what I just loved to pieces about this book in a very little nutshell. Other things of no less importance were how well-written McGuire's books remain. It's really an average-sized book, clocking in at a little over 300 pages, but you're getting a very meaty, full sandwich of a book here. With condiments and everything (code for plenty of delicious info to gnaw on). I never encountered any lagging pacing or incoherent plotting. McGuire's talent at humor keeps this one a continuously fun read and both Verity and Dominic have something to learn from one another that keep their development in the realm of satisfactory.
I had only one small concern/complaint, and that was how quickly Verity warms (albeit, admittedly, lukearm at first) to Dominic to the point that she reveals certain intel on her and her family's doings or that of the InCryptid world and peoples. While on one hand she is trying to get one of The Enemy to see things in a different light, one apart from the false teachings of The Covenant, I still couldn't believe at times what she tells him in the name of gaining his trust and understanding. And there were plenty of times that Dominic proved he wasn't worthy of said consideration – but then again this also ties into his development as a character. Still, I wish this aspect had felt as organic to me as the rest of the book did.
That being said, this was really a very minor concern for me in light of how damn good the book is overall. Upon reaching The End, I wanted to go right back to page one and start all over again – one of the few universal results of reading a book that was entertaining and just about everything a reader could ask for. McGuire continues to solidify her writing as some of my most favorite and I'm ecstatic to add another of her series onto my most favorite list. As much as I moan and groan sometimes about yet another series to keep up with, I feel no such qualms about the InCryptid books and will gladly add them to the few I even bother to pre-order. If you're a fan of urban fantasy looking for something different, I can't recommend this one enough. Make room for this one on your shelves and be sure to mark the 2nd book, Midnight Blue-Light Special (March 2013) as well.
Rating: Four and a Half Scoops
Series order:
- Discount Armageddon (March 6, 2012)
- Midnight Blue-Light Special (March 2013)

subset: commoncrawl


text:
The Berenstain Bears: Camping Adventure is an action game developed by Sega and released on GameGear.
Here you will find a list of all achievements and trophies available in the game.
Do you want to earn money creating pages just like this? Gamewise is currently looking for writers, find out more here.


text:
- Age / Gender:
- 24, Male
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- Australia
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My names guy... and um Yeah!
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LUCKY DAY FOREVER Rated 4.4821 Stars Win your happiness! Drama [S] Cascade. Rated 4.4879 Stars The end of act 5 animation for Homestuck, on mspaintadventures.com Action Jerry Rated 4.4123 Stars This is Jerry Comedy - Original Lazy Writing 2 Rated 4.4446 Stars Frog-related nonsense. Comedy - Original
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text:
Hi Mommies,
My DD stopped napping - is this just a phase?
DD is 17 weeks old and weights 12lbs. She has been a great nightime sleeper, sleeping from 7pm to 6pm with one or two feeds during the night. She is swaddled, has a soother (which she doesn't always take) and a white noise machine. We always put her in her crib sleepy but awake and she'll fall asleep on average in 15 minutes.
We were less consistent with naps, but now she won't nap at all unless we take her for a looong car ride.
The minute we try to put her down in her crib, she starts crying, even when I move up her naptime so that her WT is either 60 to 90 mins only, instead of 2 hours.
DD seems so interested in the world around her she just doesn't want to close her eyes - you can see her fight the sleepiness. But by the end of the day, she is so OT, we have been having to move her BT to 6:30 if not earlier. Not ideal as its been moving her WTs to 5:30am! PLEASE HELP.
Any mommies been through this before? Do we just ride this out and hope she'll resume her regular sleeping habits in a couple of weeks?


text:
The Star's E. Thomas McClanahan finally brings red-light cameras in Kansas City to light. (1/4, Opinion, "Revenue quest, not safety, is behind cameras").
Congratulations, fellow Kansas Citians! Your motoring safety has been compromised. On May 1, 2008, the City Council unanimously voted to move forward with the installation of red-light cameras.
Don't fool yourself about the deterrent effect of red-light cameras. Any introductory criminal justice class teaches that the best deterrent for a wrongdoing is a swift and just punishment. The inconvenience and embarrassment of being pulled over on the spot for running a red light is a far greater deterrent than a notice being sent in the mail weeks later. A "ticket in the mail" is merely one more bill to pay for the offender.
If this is really about driving safety, multiple studies have concluded that extending yellow light durations is much more effective. Will the cameras be removed in the future when red-light infractions decrease?
Start the petitions for removal now.
Jeff Weis
Kansas City


text:
News about Austria, including commentary and archival articles published in The New York Times.
Chronology of Coverage
Oct. 29, 2015
Austria announces plans to build fence along border with Slovenia, provoking Slovenian officials to repeat threats of building their own fence; flare-up threatens to derail fragile European alliance amid surge of Middle Eastern migrants; Germany has also raised concerns, saying its southern state of Bavaria has become overwhelmed by asylum seekers. MORE
Oct. 12, 2015
Austria's Social Democrats retain control of office of mayor in Vienna, fending off bid in municipal elections by far-right Freedom Party to wrest control from party, which has been in power since 1945; election was watched by observers across Europe as kind of test of whether hundreds of thousands of refugees entering Europe will find acceptance. MORE
Oct. 9, 2015
Austrian historian Stephan Templ begins one year prison term in Vienna following his conviction on charges of defrauding state; co-author of controversial 2001 book detailing seizing of Jewish properties by Nazis became embroiled in restitution case himself; supporters suggest punishment is politically motivated. MORE
Oct. 6, 2015
Supreme Court justices seem to be in agreement to reject request from American Carol P Sachs to sue Austrian railroad in federal court in California; Sachs, severely injured on train in Innsbruck, argues she should be allowed to sue railroad in American court because she bought her Eurail pass in United States. MORE
Sep. 30, 2015
Memo from Vienna; far-right Freedom Party is challenging Social Democrats' control of Austrian government, in sign of general rise in populist, anti-immigrant mood across Europe. MORE
Sep. 25, 2015
Previous immigrants to Austria volunteer at makeshift facilities, helping to process current wave of refugees; savor chance to assist and identify with people of common background, even as they continue to struggle with their own displacement and acceptance in new country. MORE
Sep. 22, 2015
Austria assumes lead in efforts to process migrants from Middle East, as quarreling Eastern European countries tighten their borders, and rest of continent falters in addressing crisis; refugees are elated by Austria's acceptance in contrast to other less welcoming environments. MORE
Sep. 20, 2015
Migrants flood Austria, with rate hitting 10,000 in a single day, after being bounced around other European countries that remain unwilling to shelter them; situation highlights infighting among European Union member states as they fail to come to consensus on plan for influx. MORE
Sep. 6, 2015
Germans gather at train station in Munich to welcome first group of what is expected to be about 8,000 migrants to arrive over weekend; arrival culminates 10 days of intense debate and emotion as Hungary sought to prevent migrants from moving through to Austria and Germany; situation highlights need for Europe to define its policies around ongoing wave of migrants fleeing Middle East and Africa. MORE
Sep. 5, 2015
Austrian government announces that thousands of migrants kept behind Hungarian border will be given entrance to Austria and Germany; move, following emergency session of top Hungarian officials, comes in response to several days of chaotic protest by migrants kept in squalid conditions at Hungary-Austria border. MORE
Sep. 5, 2015
Austrian police say same group of smugglers responsible for deaths of 71 migrants abandoned in locked truck off Austrian highway are behind abandonment of another truck one day later; in second incident, all 81 refugees were able to escape locked vehicle; six smugglers have been arrested. MORE
Sep. 4, 2015
European law enforcement officials say smuggling of migrants into Europe has bloomed into billion-dollar business that is even larger than drug or illegal weapons trades; officials continue to arrest and pursue suspects connected to deaths of 71 migrants who suffocated in sealed truck in Austria. MORE
Sep. 1, 2015
Hundreds of migrants seeking refuge from Middle East and Africa arrive in Vienna from Budapest in series of six trains and are greeted with supplies and cheers by welcoming residents; many migrants are intent on traveling on into Germany, and local officials make process of passing through Vienna and over border as easy as possible. MORE
Aug. 31, 2015
Police continue to investigate 71 deaths of migrants found on truck in Austria, as fifth person is arrested in case; authorities consider stopping cars at Austria-Hungary border to search for human cargo, but smugglers appear to suspect trap and have begun avoiding border. MORE
Aug. 30, 2015
Austrian authorities rescue three Syrian children from smugglers' small van crammed with 23 others migrants near Braunau by German border; driver is arrested and children are being treated for severe dehydration at Braunau hospital. MORE
Aug. 30, 2015
Shivani Vora reviews Park Hyatt Hotel in Vienna. MORE
Aug. 29, 2015
Authorities arrest at least four people in connection with refrigeration truck abandoned in Austria containing remains of 71 people in rear hold who apparently died of asphyxiation; incident is part of humanitarian crisis brought on by African and Middle Eastern political instability that causes thousands to flee to Europe in overloaded and unsafe transport. MORE
Aug. 28, 2015
Truck containing bodies of up to 50 smuggled migrants is found near Vienna, underscoring scope and complexity of migration crisis in Europe; European Union leaders are faced with human traffickers who are exploiting free movement between 28-nation bloc. MORE
Aug. 28, 2015
Editorial expresses hope that discovery of decomposing bodies of refugees smuggled into Europe near summit meeting of European leaders in Vienna will lead European countries to adopt much-needed compassionate policy to cope with Continent's refugee crisis; supports Austrian Foreign Min Sebastian Kurz's plan calling for quotas for each European Union member state, creation of safe havens, and crackdown on traffickers. MORE
Jul. 12, 2015
In Transit blog; H&I Adventures offers mountain bike tour that travels through parts of Slovenia, Austria and Italy in one day, focusing on local cuisine as well as cycling. MORE
May. 1, 2015
Austrian Judge Christoph Bauer refuses United States Justice Dept request to extradite Ukrainian billionaire Dmitri V Firtash to face federal racketeering and related charges in Chicago; cites insufficient against Firtash related to $18.5 billion in bribes to secure titanium-mining deal in India. MORE
Mar. 7, 2015
Austria's Art Restitution Advisory Board issues nonbinding recommendation that government keep possession of Gustav Klimt's Beethoven Frieze; wants it to remain at Vienna Secession building rather than be given to heirs of Erich Lederer, who sold it to government in 1972. MORE
Feb. 26, 2015
Austria's Parliament issues law banning foreign funding for country's Islamic organizations and requiring that Muslim groups use standardized German translation of Quran; dictate, seen as offensive by some Muslims, is grudgingly accepted. MORE
Feb. 23, 2015
Waldorf Astoria's Viennese Opera Ball celebrates 60th anniversary of the signing of the Austrian state treaty, agreement that officially ended allied occupation of Austria following World War II; event features 71-year-old Jeep that was used to patrol the occupied territory. MORE
Feb. 12, 2015
Braunau Am Inn Journal: city of Braunau Am Inn, Austria, is trying to decide what to do with vacant building where Adolph Hitler was born; city hopes to come up with use for structure that will help overcome its tainted legacy. MORE
Jan. 15, 2015
Austrian authorities are trying to determine whether they can seize house where Adolf Hitler was born; unidentified owner has not responded to offers from government to purchase house. MORE
Jan. 4, 2015
Andrew Ferren travel article on 36-hour trip to Vienna. MORE
Dec. 17, 2014
Austrian forensics experts say they will need at least two months to see if they can identify victims of apparent massacre of 43 trainee teachers in Mexico; victims were abducted by corrupt police officers, and their bodies incinerated by drug gang members. MORE
Nov. 29, 2014
Austrian police arrest 13 people suspected of recruiting fighters for radical Islamic groups in Middle East after search of homes and mosques uncovers terrorism-related propoganda material and unspecified computer data. MORE
Oct. 30, 2014
Austrian authorities say they have arrested 14-year-old boy on suspicion of planning to place bomb in a Vienna's Westbahnhof train station and may charge him with with belonging to terrorist organization. MORE
Oct. 25, 2014
Austria unveils new monument dedicated to thousands of Austrians who deserted from the German Army during World War II, marking a new step in efforts to acknowledge the nation's wartime past. MORE
Jul. 23, 2014
Austrian energy company OMV and its chief executive Gerhard Roiss find themselves in difficult position as European Union weighs tougher sanctions against Russia in wake of downing of Malaysia Airlines Flight 17; OMV had recently signed natural gas pipeline deal with Gazprom; OMV's predicament illustrates difficulties of easing energy chokehold that gives Russia so much leverage in EU. MORE
Apr. 20, 2014
Freelance journalist Stephan Templ has been convicted of defrauding the Austrian state by willfully omitting his estranged aunt Elisabeth Kretschmer from restitution claim he made in 2005; depending on one's viewpoint, case is an example of wrongful imprisonment, naked avarice or how feuds among multiple heirs can undermine justice in Holocaust restitution cases. MORE
Mar. 27, 2014
European banks are caught in financial and geopolitical crossfire as Western allies seek to impose sanctions on Russia following crisis in Crimea; are vulnerable to Russia for about $194 billion, compared with about $37 billion for American banks; problem is especially difficult for Austrian banks that pushed aggressively into the newly open markets of Eastern Europe following dissolution of the Soviet Union. MORE
Oct. 16, 2013
Heirs of Jewish art collector Erich Lederer are seeking restitution for Gustav Klimt's Beethoven Frieze, which Lederer was forced to sell to Austrian government after World War II at deep discount in order to obtain export licenses for his other artworks. MORE
Sep. 30, 2013
Austria's two biggest political parties, the Social Democrats and the Austrian People's Party, make worst showings in decades in national election, losing votes to array of smaller parties, but retaining enough seats in Parliament to stay in power. MORE
May. 23, 2013
European Union inches closer to ending bank secrecy when Austria agrees to eventually start sharing personal bank account information with other countries, as long as similar rules also apply to tax havens like Switzerland that are not part of 27-nation bloc. MORE
May. 9, 2013
Austria faces renewed pressure to reveal more about tax evaders after European Union calls for automatic information sharing to apply more widely and rapidly within bloc; lack of single tax authority has long allowed country to make tax affairs more opaque than other members. MORE
Apr. 13, 2013
Austrian Finance Min Maria Fekter says her country will not drop its banking secrecy rules, defying growing international attempt to root out tax havens on the Continent; Fekter accuses Britain of hypocrisy, noting that several tax havens like the Cayman Islands remain under its control yet outside European oversight. MORE
Mar. 27, 2013
Up to 100 vehicles collide on snow-slicked stretch of highway about 40 miles west of Vienna, leaving one person dead. MORE
Mar. 23, 2013
Saturday Profile of Rev Helmut Schuller, who has reputation as one of leading rebels within Austrian Roman Catholic Church; Schuller is one of organizers behind group of more than 400 priests and deacons who in 2011 issued 'Appeal to Disobedience,' which called for reforming church practices. MORE
Mar. 4, 2013
Team Stronach, Austrian pro-business and anti-euro political party, wins state parliament seats in Carinthia and Lower Austria, building momentum for national elections in the fall; party was founded by Austrian-Canadian auto-parts magnate Frank Stronach. MORE
Feb. 3, 2013
Evan Rail travel article on 36-hour visit to Innsbruck, Austria. MORE
Jan. 21, 2013
Nearly 60 percent of Austrian voters reject referendum seeking to change traditional system of compulsory military service for all 18-year-old men to smaller, more mobile professional force; many say that they believe that country is better served by defense filled with conscripts. MORE
Dec. 10, 2012
Expected merger of Austrian mobile carriers 3Austria and Orange Austria could set precedent that would make other telecommunications takeovers easier, potentially fulfilling a long-predicted consolidation of the industry in Europe. MORE
Dec. 9, 2012
Spectators and skiers from all over the world travel to Rettenback glacier in Solden, Austria, every October to watch first World Cup race of season and enjoy luxuriant resorts, hearty fare and the thrill of the slopes. MORE
Jul. 25, 2012
Markus Wallner, provincial governor of Austria's Vorarlberg Province, orders state-run hospitals to stop circumcising boys, wading into a religiously charged debate after a regional court in Germany banned the procedure as physical abuse. MORE
Jun. 27, 2012
Austrian fund to compensate victims of Nazi persecution will shut down after paying out $210 million for just a fraction of its recognized claims; fund cites limited finances. MORE
May. 3, 2012
Austria will join a growing list of European countries and institutions whose leaders are planning to boycott soccer championship matches in Ukraine in protest of the imprisonment of Yulia V Tymoshenko, a former prime minister; her supporters believe she has been targeted for reprisals by her main political rival, Ukraine's president, Viktor F Yanukovich. MORE
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General Information on Austria
Official Name: Republic of Austria
Capital: Vienna (Current local time)
Government Type: Federal republic
Population: 8.2 million
Area: 32,377 square miles; slightly smaller than Maine
Languages: German, Turkish, Serbian, Croatian (official in Burgenland)
GDP Per Capita: $34,700
Web site: Parlament.gv.at (In German)
Supreme Court Says Woman Injured in Austria Can't Sue in U.S.
Carol P. Sachs of California, who lost her legs after a train accident in Innsbruck, sought to sue the railroad in federal court because she bought her ticket in the United States.
December 2, 2015, Wednesday
MORE ON AUSTRIA AND: Railroad Accidents and Safety , Suits and Litigation (Civil) , Supreme Court (US) , Roberts, John G Jr , Austria
Making a Museum Out of a Mountain
In picture-perfect South Tyrol on the Austrian border of Italy, the legendary mountaineer Reinhold Messner has created six eccentric museums devoted to his lifelong passion. Naturally, to get to each one is a hike.
November 12, 2015, Thursday
Austria's Marcel Hirscher Hopes to Continue Alpine Dominance
As the 2016 World Cup season begins, Hirscher, a slalom specialist, is looking to win a record fifth consecutive World Cup overall title.
October 30, 2015, Friday
MORE ON AUSTRIA AND: Alpine Skiing , World Cup (Skiing) , Ligety, Ted , Svindal, Aksel Lund , Jansrud, Kjetil , Hirscher, Marcel , Solden (Austria)
Austria and Slovenia Trade Threats as Tensions Over Migrants Grow
The fragile alliance over migrants in Europe showed signs of strain after Austria and Slovenia threatened to build border fences and Bavaria sought to stem the flow into Germany.
October 29, 2015, Thursday
MORE ON AUSTRIA AND: Middle East and Africa Migrant Crisis , Austria , Slovenia , Germany , Fences , Refugees and Displaced Persons , European Union , Hungary
Migrant Suffocations in Truck Near Hungary Reveal Tactics of Smugglers
The deaths of 71 migrants in a meat truck found in Austria raised questions about the cruelty of the smugglers and the desperation of their clients.
October 20, 2015, Tuesday
MORE ON AUSTRIA AND: Middle East and Africa Migrant Crisis , Human Trafficking , Refugees and Displaced Persons , Smuggling , Europe , Austria , Asphyxiation, Choking, and Suffocation , Deaths (Fatalities) , Hungary
Reporter's Notebook: 71 Migrants Dead in a Truck. Why?
David D. Kirkpatrick describes his experience reporting the story of how and why the migrants willingly entered an airless meat truck in Austria.
October 20, 2015, Tuesday
MORE ON AUSTRIA AND: Middle East and Africa Migrant Crisis , Europe , Austria , Libya , Human Trafficking
1915: Servians Fight Like Lions
From the International Herald Tribune archives: Servia inflicts a heavy loss on Austro-German invaders in 1915.
October 15, 2015, Thursday
MORE ON AUSTRIA AND: Austria , Belgrade (Serbia) , Bulgaria , World War I (1914-18)
Rent a Room in Austria, Ride in a Maserati (or Jaguar)
The new Grand Ferdinand hotel is situated on Vienna's 150-year-old Ringstrasse, whose glamorous origins inspired the décor.
October 15, 2015, Thursday
MORE ON AUSTRIA AND: Hotels and Travel Lodgings , Vienna (Austria) , Austria
Vienna Looks East for Fine Art
Vienna has gained a reputation as a cultural hub for the region east of its borders -- an image that many of its arts institutions have been working to promote.
October 14, 2015, Wednesday
MORE ON AUSTRIA AND: Art , Culture (Arts) , Vienna (Austria)
Far-Right Party Gains, but Falls Short in Vienna Election Seen as Refugee Test
The Social Democrats viewed the result as a triumph for their policy of acceptance, though the Freedom Party garnered 31 percent of the vote.
October 12, 2015, Monday
MORE ON AUSTRIA AND: Middle East and Africa Migrant Crisis , Austria , Refugees and Displaced Persons , Elections , Freedom Party of Austria , Social Democratic Party (Austria) , Merkel, Angela , Haupl, Michael
Austria Weighs Building Border Fence
Along with other options, Austria is considering building a fence along its border with Slovenia to help control the influx of migrants heading north through Europe.
Austria 'Out of Order,' Germany Says
The German interior minister, Thomas de Maizière, on Wednesday criticized Vienna's handling of the migration crisis, after the Austrian government said it was considering building a border fence.
The Train to Germany
Syrian migrants took a train from Austria to their final destination: Germany.
Vienna Welcomes Refugees
Austrians received refugees with food, drinks and applause as they arrived by train from Hungary this weekend.
Migrants' Journey
Thousands of migrants arrived in Austria on Saturday morning after Hungary helped them travel in safety.
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Police arrested a township man they say had heroin and fled when confronted.
Raymond Plunkett, 37, is charged with possession of heroin, possession of heroin with intent to distribute it and resisting arrest, Lt. Troy Bezak said.
Two officers found Plunkett around 5 p.m. Wednesday while patrolling the area behind Great Bay Plaza, which is known as a popular place for graffiti and other illicit activities, Bezak said. ...