data_sheet_1_Stabilization of Hypoxia-Inducible Factor-1 Alpha Augments the Therapeutic Capacity of Bone Marrow-Derived Mesenchymal Stem Cells in Experimental Pneumonia.DOC

<p>Bone marrow-derived mesenchymal stem cells (MSCs) have therapeutic effects in experimental models of lung injury. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcriptional regulator that influences cellular metabolism, energetics, and survival under hypoxic conditions. The current study investigated the effects of stabilizing HIF-1α on the therapeutic capacity of MSCs in an experimental mouse model of bacterial pneumonia. HIF-1α stabilization was achieved by the small molecule prolyl-hydroxlase inhibitor, AKB-4924 (Aerpio Therapeutics, Inc.), which blocks the pathway for HIF-1α degradation in the proteosome. In vitro, pre-treatment with AKB-4924 increased HIF-1α levels in MSCs, reduced the kinetics of their cell death when exposed to cytotoxic stimuli, and increased their antibacterial capacity. In vivo, AKB-4924 enhanced MSC therapeutic capacity in experimental pneumonia as quantified by a sustainable survival benefit, greater bacterial clearance from the lung, decreased lung injury, and reduced inflammatory indices. These results suggest that HIF-1α stabilization in MSCs, achieved ex vivo, may represent a promising approach to augment the therapeutic benefit of these cells in severe pneumonia complicated by acute lung injury.</p

Direct anti-microbial mechanisms from neutrophils and the GAS counterattack.

<p>Neutrophils are equipped with multiple anti-infective strategies including the bacterial uptake (phagocytosis), the phagolysosomal degradation of bacteria via reactive oxygen species (oxidative burst), the release of antimicrobial molecules (degranulation), and the formation of a web-like structure composed of chromatin, histones, and antimicrobials (neutrophil extracellular traps [NETs]). GAS is equipped with a magnitude of neutrophil resistance factors (grey boxes) allowing the pathogen to uniquely counteract each anti-bacterial strategy of neutrophils.</p

Neutrophil anti-bacterial functions subverted by GAS.

<p>GAS produces a large suite of virulence factors to counteract specific neutrophil clearance mechanisms during the pathogenesis of invasive infection.</p

Key role of hypochlorite (HOCl) in triggering NETs.

<p>(A,B) Human neutrophils (2×10⁶ cells/well) were pre-treated with ABAH, and then stimulated with either H₂O₂ or NaOCl. MPO inhibition blocked H₂O₂-stimulated but not NaOCl-stimulated NETosis, as seen with (A) microscopy and (B) quantification of extracellular DNA release, indicating that HOCl is the central oxidant species that triggers NET formation in human neutrophils. All conditions were done in triplicate. (C) Stimulated human neutrophils incubated in minimal media with or without Cl⁻ were treated with exogenous H₂O₂ or NaOCl at indicated concentrations. In the absence of Cl⁻, human NETosis was rescued by addition of exogenous NaOCl, but not by H₂O₂, again illustrating that HOCl represents the central oxidant species for NET formation. All conditions were done in triplicate.</p

Human neutrophils form NETs in response to exogenous oxidants.

<p>(A) Hypochlorite (HOCl) release by activated neutrophils incubated in minimal media with or without Cl⁻ was measured using a colorimetric assay for chlorination of extracellular taurine. Production of HOCl by activated neutrophils requires extracellular Cl⁻. Each condition was done in triplicate. (B,C) NETosis increased in dose-dependent manner in response to exogenous hydrogen peroxide and sodium HOCl, as visualized using immunofluorescence and microscopy (blue: DAPI, green: anti-MPO antibody) (B), and using quantification of released extracellular DNA (C) as assays of NETosis. Neutrophils were plated at 2×10⁶ cells/well.</p

Extracellular chloride (Cl⁻) contributes to bacterial killing by human neutrophils.

<p>(A) Human peripheral neutrophils were incubated in minimal media with or without Cl⁻ for 3 h (2×10⁶ cells/well). Cells were then assayed for viability using calcein (green) which identifies live cells, and ethidium homodimer (red) which binds DNA but cannot cross intact cell membranes, thus staining dead cells. Neutrophils incubated for 3 h in minimal media with and without Cl⁻ are equally viable. (n = 3 wells per condition) (B) Human peripheral neutrophils were incubated in minimal media with or without Cl⁻. Bacteria were incubated in same media, with and without neutrophils (MOI  = 0.1). Bacterial killing by neutrophils was reduced in the absence of extracellular Cl⁻. Each condition performed in triplicate. (C) Oxidative burst was assayed using intracellular DCF dye that fluoresces when oxidized. The production of oxidants was slower in the absence of Cl⁻, but similar peak production of oxidants was achieved by 100 min in the presence or absence of Cl⁻. Each condition was done in triplicate in each experiment (D) Oxidant production in activated neutrophils occurs in a series of reactions. Enzymes known to be important in these reactions, such as NADPH oxidase and MPO are also essential for NETosis.</p

NET formation requires extracellular chloride (Cl⁻).

<p>Human peripheral neutrophils (2×10⁶ cells/well) were incubated in minimal media with or without extracellular Cl⁻, and stimulated to form NETs with PMA for 3 h. NETosis was completely inhibited in the absence of extracellular Cl⁻, as seen by (A) immunofluorescence using DAPI to counterstain DNA (blue) and anti-MPO antibody (green) to visualize NETs, and (B) quantification of released extracellular DNA (each condition done in triplicate).</p

Differing roles of myeloperoxidase (MPO) in human and murine NETosis.

<p>(A) The pharmacological agent ABAH inhibited MPO in both human, and murine bone marrow-derived neutrophils, as evidenced by significantly decreased MPO-catalyzed HOCl release after activation with PMA. All conditions were done in triplicate. (B,C) Human peripheral neutrophils were stimulated with potent trigger for NETosis (live <i>Pseudomonas aeruginosa</i> bacteria, MOI  = 1) in the presence of ABAH or vehicle control (blue: DAPI, green: anti-H2A/H2B/DNA complex antibody). Inhibition of MPO with ABAH almost completely blocked human NET formation even in response to potent stimuli (B, upper panels). However, inhibition of MPO with ABAH in murine bone marrow-derived neutrophils (B, middles panels) or skin pouch derived neutrophils (B, lower panels) did not block NET formation significantly. Data were obtained using (B) microscopy and immunofluorescence and (C) quantification of released extracellular DNA. As <i>P. aeruginosa</i> is known to release extracellular DNA itself, we included a bacteria only control, showing that bacteria alone do not significantly contribute to the elevation in released extracellular DNA. All conditions were performed in triplicate. (D,E) Bone marrow derived-neutrophils from MPO knockout mice or wild type siblings were stimulated for indicated times with live <i>P. aeruginosa</i> bacteria (MOI  = 1). No differences in NET formation were observed with either direct visualization with (D) immunofluorescence and microscopy or (E) quantification of released extracellular DNA. All conditions done in triplicate, experiment repeated two independent times.</p

Stimulation of murine macrophages by human and GAS DNA.

<p>(<b>A–C</b>) Bone marrow-derived mice macrophages (BMDMs) were stimulated with GAS and human genomic DNA (both 5 µg/ml) and secretion of IFN1, IFN-α and TNF-α in the supernatants measured. (<b>D</b>) Dose-dependency of GAS DNA-mediated stimulation (12 hours) of IFN-α and TNF-α secretion. Data were pooled from 3 experiments done in triplicates and presented as mean ± SEM. * <i>P</i><0.05 ** <i>P</i><0.01.</p

The GAS DNase Sda1 interferes with TLR9 activation.

<p>(<b>A</b>) IFN-α and TNF-α secretion by BMDMs after GAS DNA challenge for 12 hours was almost abolished after adding recombinant Sda1 to GAS DNA. (<b>B</b> and <b>C</b>) Stimulation of BMDMs by GAS strains expressing Sda1 (GASM1 WT and GASM49 pD<i>csda1</i>) for 12 hours secreted significantly less IFN-α and TNF-α compared to matching strains lacking Sda1 (GASM1 Δ<i>sda1</i> and GASM49 WT pD<i>cerm</i>). Data were pooled from 3 experiments done in triplicates and presented as mean ± SEM. ** <i>P</i><0.01 *** <i>P</i><0.001.</p