Endothelial ATP release during hypoxia.

<p>A, To study extracellular ATP release from normoxic endothelia, monolayers of confluent HMEC-1 were washed and the culture media was replace with calcium containing HBSS. ATP content from their supernatant was sampled at indicated time points and quantified using a luminometric ATP detection assay. B, To measure extracellular ATP release under hypoxic conditions, confluent HMEC-1 monolayers were exposed to hypoxia (2% oxygen) over indicated time periods. Culture media was replaced with calcium containing HBSS and the ATP content within the supernatant was measured after 30 min incubation time. C, For intracellular ATP measurement, confluent HMEC-1 monolayers were exposed to hypoxia over indicated time periods, culture medium was discarded and cells were lysed by adding ice-cold water. ATP concentrations were measured as above. D, To measure the influence of different oxygen concentrations on endothelial ATP release, HMEC-1 were exposed to indicated degrees of hypoxia (21–2% of oxygen) over 24 h. Culture media was replaced with calcium containing HBSS and the ATP content within the supernatant was measured after 30 min incubation time. E, Confluent HMEC-1 monolayers were exposed to hypoxia as indicated. To assess lytic ATP release, LDH concentrations within the supernatant were measured by an LDH detection kit. In control experiments, cells were lysed with Triton X-100 (*p<0.01, n = 6 for all experiments).</p

Endothelial connexin expression.

<p>Confluent HMEC-1 monolayers were exposed to normoxia or hypoxia (12 h). Total RNA was isolated and real-time reverse-transcriptase polymerase chain reaction was employed to screen for transcriptional modulation of connexin expression. Data were calculated relative to an internal control gene (β-actin) and are expressed as fold change over normoxia at each time point. Results are derived from 3 experiments in each condition.</p

Influence of hypoxia on Connexin 43 expression.

<p>A, B. Confluent HMEC-1 or HUVEC monolayers were exposed to normoxia or hypoxia (2% oxygen) over indicated time periods. Total RNA was isolated and transcriptional responses were assessed by real-time reverse-transcriptase polymerase chain reaction. Data were calculated relative to an internal housekeeping gene (β-actin) and are expressed as fold change over normoxia at each time point. Results are derived from 3 experiments in each condition. C. Confluent HMEC-1 monolayers were exposed to hypoxia over indicated time periods. Cells were lysed and proteins were resolved by SDS-PAGE and transferred to PVDF-membrane. Membranes were probed with a connexin 43 antibody, proteins were detected by chemiluminescene. The same blot was reprobed for β-actin as a control for protein loading. A representative experiment of 3 is shown. D. Confluent HMEC-1 monolayers were exposed to hypoxia over indicated time periods. Monolayers were washed, surface proteins were biotinylated, and cells were lysed. Connexin 43 was immunoprecipitated, followed by addition of Protein G Microbeads. Proteins were resolved by SDS-PAGE and resultant Western blots were probed with Streptavidin. A representative experiment of 3 is shown. E. Confluent HMEC-1 monolayers were exposed to normoxia or hypoxia over indicated time periods. Cells were lysed and proteins were resolved by SDS-PAGE and transferred to PVDF-Membrane. Membranes were probed with phospho-connexin 43 antibody specific for phosphorylated ser368, and proteins were detected by chemiluminescene. The same blot was probed for β-actin as a control for protein loading. A representative of 3 is shown. In subsets of experiments, cells were pretreated with the protein kinase C inhibitor bisindolylmaleimide (10 µM; +BIM).</p

Connexin-mimetic peptides in endothelial ATP release.

<p>A, B, Confluent HMEC-1 monolayers were washed and treated with connexin-mimetic peptides (A: Cx40 peptide, SRPTEKNVFIV, 50 µmol; B: Cx43 peptide, SRPTEKTIFII, 50 µmol). ATP content within the supernatant was measured by a luminometric ATP detection assay after an incubation period of 20 min and compared with control HMEC-1 treated with 50 µM bovine albumin (n = 6).</p

ATP release from HMEC treated with the protein kinase C inhibitor bisindolylmaleimide (BIM).

<p>To study the role of Cx43 ser398 phosphorylation status in ATP release from endothelia, monolayers of confluent HMEC-1 were treated with BIM (+BIM, 10 µM) or vehicle control (-BIM), exposed to normoxia or hypoxia (24 h, 2% oxygen), washed and the culture media was replace with calcium containing HBSS. ATP content from the supernatant was sampled at indicated time points and quantified using a luminometric ATP detection assay (*p<0.05 compared to Normoxia – BIM; n = 6).</p

Human primer pairs used for real-time RT-PCR.

<p>Human primer pairs used for real-time RT-PCR.</p

Molecular mechanisms of endothelial-dependent ATP release.

<p>A–D, Confluent HMEC-1 monolayers were washed and exposed to Verapamil (10 µmol) or Dipyridamole (1 µmol) over 20 min, 18-alpha-glycyrrhetinic acid (18αGA, 20 µmol) or Anandamide (40 µmol) over 10 min. ATP content within the supernatant was measured by a luminometric ATP detection assay and compared with untreated control cells (n = 6).</p

Role of Arginase 1 from Myeloid Cells in Th2-Dominated Lung Inflammation

<div><p>Th2-driven lung inflammation increases Arginase 1 (Arg1) expression in alternatively-activated macrophages (AAMs). AAMs modulate T cell and wound healing responses and Arg1 might contribute to asthma pathogenesis by inhibiting nitric oxide production, regulating fibrosis, modulating arginine metabolism and restricting T cell proliferation. We used mice lacking Arg1 in myeloid cells to investigate the contribution of Arg1 to lung inflammation and pathophysiology. In six model systems encompassing acute and chronic Th2-mediated lung inflammation we observed neither a pathogenic nor protective role for myeloid-expressed Arg1. The number and composition of inflammatory cells in the airways and lungs, mucus secretion, collagen deposition, airway hyper-responsiveness, and T cell cytokine production were not altered if AAMs were deficient in Arg1 or simultaneously in both Arg1 and NOS2. Our results argue that Arg1 is a general feature of alternative activation but only selectively regulates Th2 responses. Therefore, attempts to experimentally or therapeutically inhibit arginase activity in the lung should be examined with caution.</p></div

Specific Microbiome Changes in a Mouse Model of Parenteral Nutrition Associated Liver Injury and Intestinal Inflammation

<div><p>Background</p><p>Parenteral nutrition (PN) has been a life-saving treatment in infants intolerant of enteral feedings. However, PN is associated with liver injury (PN Associated Liver Injury: PNALI) in a significant number of PN-dependent infants. We have previously reported a novel PNALI mouse model in which PN infusion combined with intestinal injury results in liver injury. In this model, lipopolysaccharide activation of toll-like receptor 4 signaling, soy oil-derived plant sterols, and pro-inflammatory activation of Kupffer cells (KCs) played key roles. The objective of this study was to explore changes in the intestinal microbiome associated with PNALI.</p><p>Methodology and Principal Findings</p><p>Microbiome analysis in the PNALI mouse identified specific alterations within colonic microbiota associated with PNALI and further association of these communities with the lipid composition of the PN solution. Intestinal inflammation or soy oil-based PN infusion alone (in the absence of enteral feeds) caused shifts within the gut microbiota. However, the combination resulted in accumulation of a specific taxon, <i>Erysipelotrichaceae</i> (23.8% vs. 1.7% in saline infused controls), in PNALI mice. Moreover, PNALI was markedly attenuated by enteral antibiotic treatment, which also was associated with significant reduction of <i>Erysipelotrichaceae</i> (0.6%) and a Gram-negative constituent, the S24-7 lineage of Bacteroidetes (53.5% in PNALI vs. 0.8%). Importantly, removal of soy oil based-lipid emulsion from the PN solution resulted in significant reduction of <i>Erysipelotrichaceae</i> as well as attenuation of PNALI. Finally, addition of soy-derived plant sterol (stigmasterol) to fish oil-based PN restored <i>Erysipelotrichaceae</i> abundance and PNALI.</p><p>Conclusions</p><p>Soy oil-derived plant sterols and the associated specific bacterial groups in the colonic microbiota are associated with PNALI. Products from these bacteria may directly trigger activation of KCs and promote PNALI. Furthermore, the results indicate that lipid modification of PN solutions may alter specific intestinal bacterial species associated with PNALI, and thus suggest strategies for management of PNALI.</p></div

Arg1 expression by macrophages does not regulate acute schistosome egg-induced pulmonary granuloma formation and Th2 response.

<p>Mice deficient in Arg1, iNOS, or both enzymes were sensitized by i.p. injection of eggs, then challenged by a single i.v. injection of eggs to form pulmonary granulomas and analyzed at 2 weeks. A) T lymphocytes (CD3+), macrophages (F4/80+), and Arginase1+ cells were detected in fixed lung serial sections using immunohistochemistry. Images represent 8 mice of each genotype. B) The volume and eosinophil composition of 10–30 granulomas per mouse were scored in Geimsa-stained lung sections. C) Collagen deposition was compared by measuring L-hydroxyproline in matched lobes and calculating, by mass, total lung content. D) Expression of Arginase 2 or E) IL-13 mRNA and the Th2-responsive genes RELM-α, Mucin 5AC, and Gob5 were compared in lung tissue by quantitative PCR, normalized to the mean naïve WT level. F) To evaluate CD4 T cell responses, lung leukocytes were restimulated with PMA plus ionomycin and stained to detect IL-13, IL-4, IL-5, and IFN-γ by flow cytometry. No significant differences were observed between the challenged groups in B) to F). Results were combined from two independent experiments totaling 11 to 19 mice per group, plus 6 naïve WT controls. Individual mice and group means are shown.</p