Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 1. Pulmonary and plasma osteopontin concentrations are elevated during pneumococcal pneumonia. Osteopontin concentrations in (A) lung and (B) plasma before and 6, 24, and 48 h after infection with 104 colony-forming units of Streptococcus pneumoniae. Data are expressed as mean 6 standard error of the mean (SEM); n 5 8 mice per group. Asterisk, P , .05; double asterisk, P , .01; triple asterisk, P , .001, compared with t 5 0. Osteopontin concentrations in culture supernatants after incubation of (C) MH-S cells and (D) primary alveolar macrophages with medium or growth- arrested S. pneumoniae (multiplicity of infection, 1:6 and 1:60 for MH-S cells; 1:20 and 1:200 for primary alveolar macrophages) for 4 h (MH-S cells) or 20 h (primary alveolar macrophages). Data are expressed as mean 6 SEM; n 5 3 per group. Asterisk, P ,.05, compared with medium. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Clinical Proteomics, Infection, Incubation

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 2. Prolonged survival and reduced bacterial growth in osteopontin knockout (KO) mice. A, Percentage survival of wild-type (WT) mice (filled symbols) and osteopontin KO mice (open symbols) after intranasal infection with 104 colony-forming units (CFU) of Streptococcus pneumoniae (n 5 14 mice per group). P value indicates the difference between groups. WT (gray) and osteopontin KO (white) mice were infected with 104 CFU of S. pneumoniae, and bacterial loads were determined 6, 24, and 48 h after infection in (B) lung, (C) blood, and (D) spleen. Data are expressed as box-and- whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group; asterisk, P ,.05; double asterisk, P ,.01; triple asterisk, P ,.001, compared with WT mice. Note to panels C and D: none of the mice in either group displayed positive blood or spleen culture results 6 h after infection; at 24 h, S. pneumoniae could be cultured from samples of the blood of only 3 of 8 osteopontin KO mice, compared with 7 of 8 WT mice and from the spleen tissue of only 1 of 7 osteopontin KO mice, compared with 7 of 8 WT mice (P , .05 and P , .01, respectively). OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Infection, Whisker Assay, Cell Culture

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 3. Decreased lung histopathology in osteopontin knockout (KO) mice. Representative lung histology of wild-type (WT) (A, D, G) and osteopontin KO (B, E, H ) mice at 6 h (A–C ), 24 h (D–F ), and 48 h (G–I) after intranasal infection with 104 CFU of Streptococcus pneumoniae. The lung sections are representative for 8 mice per group per time point. Hematoxilin and eosin staining, original magnification, 310. Inflammation scores are expressed as mean 6 standard error of the mean (WT mice, black bars; osteopontin KO mice, white bars; n 5 8 mice per group). Double asterisk, P ,.01, compared with WT mice. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Histopathology, Knock-Out, Infection, Staining

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 4. Osteopontin stabilizes Streptococcus pneumoniae viability in vitro. A, S. pneumoniae in saline (106 colony-forming units [CFU]/mL) was incubated with increasing doses (0.8–800 ng/mL) of recombinant osteopontin (black symbols) or saline (white symbols), and the viability of S. pneumoniae was determined over 6 h at 37C. B, S. pneumoniae in saline (106 CFU/mL) was incubated with 800 ng/mL recombinant osteopontin (filled squares), 800 ng/mL boiled recombinant osteopontin (open squares), 800 ng/mL bovine serum albumin (triangles), or saline (circles), and the viability of S. pneumoniae was determined over 6 h at 37C. Dashed lines depict detection limits. C, Osteopontin binds to S. pneumoniae. Enzyme-linked immunosorbent assay plates were coated or not coated with 1 3 108 CFU/mL S. pneumoniae type 3 (ATCC 6303) or serotype 2 (D39); coating with anti-osteopontin IgG was used as positive control. Binding was assessed using biotin-labeled recombinant mouse osteopontin. Data are means 6 standard error (n 5 4–6). Double asterisk, P , .01 vs buffer. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: In Vitro, Saline, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Binding Assay, Labeling

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 5. Similar bacterial growth during pneumococcal sepsis. Bacterial loads in (A) blood, (B) lung, (C) liver, and (D) spleen from wild-type (WT; gray) and osteopontin knockout (OPN KO; white) mice at 24 and 48 h after intravenous injection with 105 colony-forming units (CFU) of Streptococcus pneumoniae. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group. Dashed line depicts detection limit.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Injection, Whisker Assay

Journal: Frontiers in Immunology

Article Title: Osteopontin from monocyte-derived dendritic cells mediates ozone-induced pulmonary responses in mice

doi: 10.3389/fimmu.2026.1748667

Figure Lengend Snippet: Cell counts in bronchoalveolar lavage fluid (BALF), airway hyperresponsiveness, and concentrations of osteopontin in lungs of mice exposed to air or ozone. (A) Experimental protocol. (B) Results of cell counts in BALF in mice exposed to air or ozone. (C) Lung histological examination by hematoxylin and eosin staining. (D) Results of airway hyperresponsiveness in mice exposed to air or ozone. (E) Concentrations of osteopontin in mice exposed to air or ozone. The data shown are pooled from multiple experiments (n = 5–7 per group). Results are means ± standard error. **p < 0.01, *p < 0.05 compared with mice exposed to air.

Article Snippet: In addition, 1.0x10 6 bone marrow neutrophils were stimulated with 1.0 μg/mL of mouse recombinant osteopontin (R&D Systems) in a 24-well dish for 2 hours, referring to a previous report , and collected for further RNA extraction and flowcytometric analyses.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Osteopontin from monocyte-derived dendritic cells mediates ozone-induced pulmonary responses in mice

doi: 10.3389/fimmu.2026.1748667

Figure Lengend Snippet: Cell counts of monocyte-derived dendritic cells and macrophages in lungs, osteopontin concentrations in lungs, cell counts in bronchoalveolar lavage fluid (BALF), and airway hyperresponsiveness in mice treated with control liposome or clodronate liposome and exposed to air or ozone. (A) Experimental protocol. (B) Flowcytometric analysis of single lung cells in mice treated with control liposome or clodronate liposome. (C, D) Ratios of moDCs and macrophages in lungs of mice treated with control liposome or clodronate liposome. (E) Concentrations of osteopontin in lungs of mice treated with control liposome or clodronate liposome and exposed to air or ozone. (F) Cell counts in BALF and (G) airway hyperresponsiveness in mice treated with control liposome or clodronate liposome and exposed to air or ozone. The data shown are pooled from multiple experiments (n = 4–6 per group). Results are means ± standard error. *p < 0.05, **p < 0.01 compared with mice exposed to air. †p < 0.05, ††p < 0.01 compared with mice exposed to ozone and treated with control liposome. FCM, flow cytometry; FSC, forward scatter; SSC, side scatter; moDC, monocyte-derived dendritic cell; cont.lipo, control liposome; CL.lipo, clodronate liposome.

Article Snippet: In addition, 1.0x10 6 bone marrow neutrophils were stimulated with 1.0 μg/mL of mouse recombinant osteopontin (R&D Systems) in a 24-well dish for 2 hours, referring to a previous report , and collected for further RNA extraction and flowcytometric analyses.

Techniques: Derivative Assay, Control, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Osteopontin from monocyte-derived dendritic cells mediates ozone-induced pulmonary responses in mice

doi: 10.3389/fimmu.2026.1748667

Figure Lengend Snippet: Cell counts in bronchoalveolar lavage fluid (BALF) and airway hyperresponsiveness in mice treated with placebo or osteopontin inhibitor and exposed to air or ozone. (A) Experimental protocol. (B) Cell counts in BALF of mice treated with placebo or osteopontin inhibitor and exposed to air or ozone. (C) Airway hyperresponsiveness in mice treated with placebo or osteopontin inhibitor and exposed to air or ozone. The data shown are pooled from multiple experiments (n = 4–6 per group). Results are means ± standard error. *p < 0.05, **p < 0.01 compared with mice exposed to air. ††p < 0.01 compared with mice exposed to ozone and treated with placebo.

Article Snippet: In addition, 1.0x10 6 bone marrow neutrophils were stimulated with 1.0 μg/mL of mouse recombinant osteopontin (R&D Systems) in a 24-well dish for 2 hours, referring to a previous report , and collected for further RNA extraction and flowcytometric analyses.

Techniques:

Journal: Frontiers in Immunology

Article Title: Osteopontin from monocyte-derived dendritic cells mediates ozone-induced pulmonary responses in mice

doi: 10.3389/fimmu.2026.1748667

Figure Lengend Snippet: RNA sequencing and protein analyses of bone marrow neutrophils stimulated by osteopontin. (A) Experimental protocol of purification of bone marrow neutrophils. (B) Morphological analysis of bone marrow neutrophils by Diff-Quik staining. (C) Principal component scores with the confidence ellipsoids in bone marrow neutrophils with or without stimulation by osteopontin. (D) Volcano plots for differential expression analysis in bone marrow neutrophils with or without stimulation by osteopontin. (E) Gene expression of NDUFA1 in bone marrow neutrophils with or without stimulation by osteopontin. The data shown are pooled from 6 samples per group. Results are means ± standard error. *p < 0.05 compared with the result of bone marrow neutrophils without stimulation.

Article Snippet: In addition, 1.0x10 6 bone marrow neutrophils were stimulated with 1.0 μg/mL of mouse recombinant osteopontin (R&D Systems) in a 24-well dish for 2 hours, referring to a previous report , and collected for further RNA extraction and flowcytometric analyses.

Techniques: RNA Sequencing, Purification, Diff-Quik, Staining, Quantitative Proteomics, Gene Expression

Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 7 iOPN was downregulated by IFN-γ-induced activation of STAT1 in MSCs. a and b Murine MSCs were treated with IFN-γ or TNF-α at the indicated concentration for 24 h. mRNA and protein of MSCs were collected. OPN expression was determined at the mRNA and protein levels by quantitative real-time PCR and immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 7a. c Neutralizing antibodies against IFN-γ, TNF-α or both were added to the medium of MSCs stimulated with IFN-γ or TNF-α. OPN expression was determined by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 7c. d–e The STAT1 inhibitor fludarabine (Flu, 2 μM) was added to the culture medium of MSCs with TNF-α plus IFN-γ (10 ng/mL) for 24 h. The expression of OPN and phosphorylation of STAT1 at Tyr701 were determined by immunoblotting analysis (d). Full-length blots are presented in Additional file 1: Fig. 7d. The mRNA expression of OPN was determined by quantitative real-time PCR (e). f iOPN luciferase activity in 293 T cells transfected with luciferase reporter and indicated expression vectors. g Schematic representation showing a conserved STAT1-binding motif located in the promoter region of OPN genes between − 2,895 and − 2,910 bp. h Enrichment of p-STAT1 at the promoter of OPN was analyzed by ChIP-PCR. i Working model of the regulation of iOPN on the immunosuppressive capacity of MSCs. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. ns = no significance

Article Snippet: Antibodies against OPN (CAT# AF808, RRID:AB_2194992) and OPN neutralizing antibodies (CAT# 441-OP) were purchased from R&D Systems (Minnesota, USA).

Techniques: Activation Assay, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Luciferase, Activity Assay, Transfection, Binding Assay