Journal: bioRxiv

Article Title: Bacterial Polyphosphates Induce CXCL4 and Synergize with Complement Anaphylatoxin C5a in Lung Injury

doi: 10.1101/2022.07.05.498838

Figure Lengend Snippet: ( A ) CXCL4 in supernatants of bone marrow derived macrophages (BMDMs) from C57BL/6J wild type mice after incubation with long-chain polyphosphates (L-PolyP; P i 700, 50 μM) or short-chain polyphosphates (S-PolyP; P i 70, 50 μM) for 24 h. Resting macrophages served as controls (Ctrl). ( B ) Dose-response of CXCL4 release from macrophages (BMDMs) with different concentrations of long-chain or short-chain polyphosphates compared to basal levels from control macrophages, 24 h. ( C ) Time course of CXCL4 release from peritoneal elicited macrophages (PEMs) after long-chain polyphosphates (50 μM). ( D ) Long-chain polyphosphates (50 μM) were incubated overnight at 37°C with recombinant exopolyphosphatase-Fc fusion protein (PPX-Fc), mutated/dead exopolyphosphatase-Fc protein (dPPX-Fc), or heat inactivated exopolyphosphatase-Fc protein (hiPPX-Fc) followed by transfer to macrophages (PEMs) and CXCL4 detection after 24 h. ( E ) Polyphosphate polymers of narrow chain length distributions were incubated with macrophages (PEMs) followed by CXCL4 detection after 24 hours. ( F ) CXCL4 induced by long-chain polyphosphates in macrophages (BMDMs) from wild type mice compared to P2Y1 -/- mice. CXCL4 was measured by ELISA for all experiments shown. Polyphosphate concentrations were 50 μM in all experiments except for frame B. All data are representative of 3 independent experiments. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The following substances were slowly injected intra-tracheally (i.t.) in 40 μl phosphate buffered saline (PBS): Synthetic polyphosphates of the doses and chain-lengths as indicated in the figure legends, recombinant mouse C5a (500ng/mouse; R&D Systems, Minneapolis, MN, USA), and recombinant mouse CXCL4 (500ng/mouse; R&D Systems, Minneapolis, MN, USA).

Techniques: Derivative Assay, Incubation, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bacterial Polyphosphates Induce CXCL4 and Synergize with Complement Anaphylatoxin C5a in Lung Injury

doi: 10.1101/2022.07.05.498838

Figure Lengend Snippet: ( A ) CXCL4 release from C57BL/6J wild type macrophages (PEMs) after incubation with long-chain or short-chain polyphosphates ± LPS (100 ng/ml), Ctrl: resting control cells, 24h, ELISA. ( B ) CXCL4 mRNA expression in macrophages (PEMs) after polyphosphates ± LPS, 24h, RT-PCR. ( C ) CXCL4 release from macrophages (BMDMs) of wild type (WT), MyD88 -/- and TRIF -/- mice, 24h, ELISA. ( D ) Relative quantification of phosphorylated Akt (threonine 308) in macrophages (BMDMs) at 0-60 min after long-chain polyphosphates. The values of fluorescence intensities (FI) were normalized to controls (0 min), bead-based assay. ( E ) CXCL4 release from polyphosphate-stimulated macrophages (BMDMs) co-treated with the Akt inhibitor, Wortmannin, 24h, ELISA. ( F ) CXCL4 release from macrophages (BMDMs) co-treated with the Akt inhibitor, Ly294002 (stock was dissolved in DMSO), 24h, ELISA. ( G ) Contour plots of phospho-Akt in F4/80 + macrophages (BMDMs) after activation with short/long-chain polyphosphates and LPS, 60 min, flow cytometry. (H) Geometric mean fluorescence intensities (gMFI) of pooled data (n=4/condition) as in frame G. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001: n.d.: not detectable.

Article Snippet: The following substances were slowly injected intra-tracheally (i.t.) in 40 μl phosphate buffered saline (PBS): Synthetic polyphosphates of the doses and chain-lengths as indicated in the figure legends, recombinant mouse C5a (500ng/mouse; R&D Systems, Minneapolis, MN, USA), and recombinant mouse CXCL4 (500ng/mouse; R&D Systems, Minneapolis, MN, USA).

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, Fluorescence, Bead-based Assay, Activation Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Bacterial Polyphosphates Induce CXCL4 and Synergize with Complement Anaphylatoxin C5a in Lung Injury

doi: 10.1101/2022.07.05.498838

Figure Lengend Snippet: ( A ) Lung sections of C57BL/6J wild type mice obtained 8h after intra-tracheal (i.t.) administration of long-chain polyphosphates (40μl at 20 mM/mouse). Sham control mice (Ctrl) received PBS (40 μl/mouse, i.t.), H&E staining, scale bar: 20 μm. ( B ) Total protein in bronchoalveolar lavage fluids (BALF) after long-chain polyphosphates or sham control treatment, 8h, BCA assay. ( C ) Representative contour plots of Ly6G + polymorphonuclear neutrophils (PMN), CD11c + SiglecF + alveolar macrophages and SiglecF + Ly6G - CD11c - eosinophils in BALF after polyphosphate-induced lung injury compared to sham controls, 8h, flow cytometry. ( D - E ) PMN frequencies and absolute numbers from mice as in frame C. ( F - G ) Frequencies and absolute numbers of alveolar macrophages (AMs) from mice as in frame C. ( H ) CXCL4 in BALF of mice in frame C. ( I ) Albumin in BALF of wild type mice administered i.t. with long-chain (L), medium-chain (M), short-chain (S) polyphosphates, or sham, 8h, ELISA. ( J ) PMN numbers in BALF from mice in frame I, manual count. ( K ) CXCL4 in BALF from C57BL/6J wild type mice as described in frame I. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001.

Article Snippet: The following substances were slowly injected intra-tracheally (i.t.) in 40 μl phosphate buffered saline (PBS): Synthetic polyphosphates of the doses and chain-lengths as indicated in the figure legends, recombinant mouse C5a (500ng/mouse; R&D Systems, Minneapolis, MN, USA), and recombinant mouse CXCL4 (500ng/mouse; R&D Systems, Minneapolis, MN, USA).

Techniques: Control, Staining, BIA-KA, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Bacterial Polyphosphates Induce CXCL4 and Synergize with Complement Anaphylatoxin C5a in Lung Injury

doi: 10.1101/2022.07.05.498838

Figure Lengend Snippet: ( A ) Albumin in BALF of C57BL/6J wild type mice after long-chain polyphosphate-induced lung injury (40μl at 20 mM/mouse) ± recombinant mouse C5a (100 ng/mouse i.t.), 8h, ELISA. ( B ) CXCL4 in BALF from mice in frame A, ELISA. ( C ) C5aR1 histograms pre-gated on PMNs or alveolar macrophages (AMs) from mice after polyphosphate-induced lung injury or sham treated controls. ( D - E ) C5aR1 surface expression as geometric mean fluorescence intensities on PMNs and AMs from mice in frame C. ( F ) Alveolar albumin in wild type (WT) and C5aR1 -/- mice after long-chain polyphosphate-induced lung injury (40μl at 20 mM/mouse), 8h, ELISA. ( G ) CXCL4 in BALF from WT and C5aR1 -/- mice in frame F, 8h, ELISA. Data are presented as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The following substances were slowly injected intra-tracheally (i.t.) in 40 μl phosphate buffered saline (PBS): Synthetic polyphosphates of the doses and chain-lengths as indicated in the figure legends, recombinant mouse C5a (500ng/mouse; R&D Systems, Minneapolis, MN, USA), and recombinant mouse CXCL4 (500ng/mouse; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence