escherichia coli  (ATCC)

Journal: Frontiers in Immunology

Article Title: Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury

doi: 10.3389/fimmu.2022.980733

Figure Lengend Snippet: Polyphosphates induce CXCL4 release from macrophages. (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, 24 h. (F) CXCL4 induced by long-chain polyphosphates in macrophages (BMDMs) of wild type mice and P2Y1 -/- mice, 24 h. 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 intratracheally (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: Frontiers in Immunology

Article Title: Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury

doi: 10.3389/fimmu.2022.980733

Figure Lengend Snippet: Polyphosphates regulate CXCL4 through PI3K/Akt signaling in macrophages. (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, 24 h, ELISA. (B) CXCL4 mRNA expression in macrophages (PEMs) after polyphosphates ± LPS, 24 h, RT-PCR. (C) CXCL4 release from macrophages (BMDMs) of wild type (WT), MyD88 -/- and TRIF -/- mice, 24 h, 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, 24 h, ELISA. (F) CXCL4 release from macrophages (BMDMs) co-treated with the Akt inhibitor, Ly294002 (stock was dissolved in DMSO), 24 h, 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 intratracheally (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: Frontiers in Immunology

Article Title: Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury

doi: 10.3389/fimmu.2022.980733

Figure Lengend Snippet: Long-chain polyphosphates cause lung injury. (A) Lung sections of C57BL/6J wild type mice obtained 8 h after intratracheal (i.t.) administration of long-chain polyphosphates (40 μl 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, 8 h, BCA assay. (C) Polyphosphate-induced lung injury in wild type mice compared to sham controls, 8 h. Representative contour plots of Ly6G + polymorphonuclear neutrophils (PMNs), CD11c + SiglecF + alveolar macrophages and SiglecF + Ly6G - CD11c - eosinophils in BALF samples are shown, 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 as 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 controls, 8 h, ELISA. (J) PMN numbers in BALF from mice as in frame I, manual count. (K) CXCL4 in BALF from 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 intratracheally (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: Frontiers in Immunology

Article Title: Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury

doi: 10.3389/fimmu.2022.980733

Figure Lengend Snippet: Polyphosphates synergize with C5a in lung injury. (A) Albumin in BALF of C57BL/6J wild type mice after long-chain polyphosphate-induced lung injury (40 μl at 20 mM/mouse) ± recombinant mouse C5a (100 ng/mouse i.t.), 8 h, ELISA. Sham control mice (Ctrl) received PBS (40 μl/mouse i.t.). (B) CXCL4 in BALF from mice as in frame A, ELISA. (C) C5aR1 histograms pre-gated on PMNs or alveolar macrophages (AMs) from representative 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 as in frame C. (F) Alveolar albumin in wild type (WT) and C5aR1 -/- mice after long-chain polyphosphate-induced lung injury (40 μl at 20 mM/mouse), 8 h, ELISA. (G) CXCL4 in BALF from WT and C5aR1 -/- mice as in frame F, 8 h, ELISA. (H) Western blots of immunoprecipitated C5a from BALF and lung lysates obtained 6 h after long-chain polyphosphate-induced lung injury (n=5) or sham treatment (Ctrl; n=4) in WT mice. Recombinant mouse C5a (~9 kDa, 10 ng) was directly applied to the lane as a positive control (Pos). The negative control (Neg) contained Protein A agarose beads and Laemmli buffer. (I, J) C5a abundance in BALF and lung as expressed by densitometry of bands shown in frame H, normalized to sham treatment. 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 intratracheally (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, Control, Expressing, Fluorescence, Western Blot, Immunoprecipitation, Positive Control, Negative Control