Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: (A) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab). At indicated post-infection time-periods the medium supernatant was collected to assess levels of mouse IL-6 by ELISA. ( B ) Primary bone marrow derived macrophages (BMDM) isolated from wild-type (WT) and S100A9 knockout (KO) mice were infected with IAV (2 MOI). The medium supernatant was collected to assess levels of mouse IL-6 by ELISA. ( C ) WT and S100A9 KO BMDM were infected with IAV (2 MOI). At the indicated post-infection time-period, medium supernatant was collected to assess levels of mouse TNF-α(TNF) by ELISA. ( D ) S100A9 KO BMDMs were infected with IAV (2 MOI) in the presence of purified recombinant mouse S100A9 protein (5 µg/ml). Medium supernatant was collected from infected cells to assess levels of mouse TNF and IL-6 by ELISA. Vehicle control cells (veh) were incubated with HBSS buffer. The values represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test.

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Isolation, Knock-Out, Purification, Recombinant, Incubation, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Mouse J774A.1 macrophages were incubated with purified recombinant mouse S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic state of these cells was examined by FACS analysis of annexin V and PI stained cells. Apoptosis rate (% apoptosis) was calculated based on number of annexin V positive/PI negative cells (denoting early apoptosis)+number of annexin V positive/PI positive cells (denoting late apoptosis)/total number of cells. ( B ) Mouse alveolar macrophage MH-S cell-line was incubated with purified S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic status was determined as described in (A). ( C ) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab). At 48 h post-infection, the apoptotic state of these cells was determined as described in (A). The values (i.e. annexin V and PI staining quantified by FACS) represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer (vehicle control).

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Incubation, Purification, Recombinant, Staining, Infection, Blocking Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Survival of IAV infected (1×10 5 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). The data represents values from two independent experiments performed with 5 mice/group for each experiment (total 10 mice/group from two experiments); *p = 0.03. ( B ) Hematoxylin and eosin (H&E) staining of lung sections from mock infected or IAV infected mice (3×10 4 pfu/mouse via intra-tracheal route) administered with either control IgG (IgG) or S100A9 Ab (24 h prior to IAV inoculation, 2 mg of antibody/mouse was administered via i.p route). Magnification, ×10. ( C ) Mice were administered with purified recombinant mouse S100A9 protein (15 µg/mouse) via intra-tracheal route. At 8 h post-administration, levels of mouse TNF-α in the lung was assessed by performing ELISA analysis with lung homogenate. ( D ) Lung homogenate prepared from mock infected and IAV infected (2×10 4 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route) were subjected to ELISA analysis to determine levels of mouse TNF-α in the lung. ( E ) For ex-vivo experiment, broncho-alveolar lavage fluid (BALF) was collected (at 3 d post-infection) from IAV infected mice (2×10 4 pfu/mouse via intra-tracheal route) administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). The BALF cells were isolated and plated in 48-well plate. After 2 h and 4 h, the medium supernatant was analyzed for mouse TNF-α (TNF) and mouse IL-6 by ELISA. Values shown in (C), (D) and (E) represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test. Veh; HBSS buffer diluted in PBS (vehicle control).

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, Staining, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Ex Vivo, Isolation, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Lung sections were prepared (at 3 d post-infection) from IAV infected (2×10 4 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). For each experimental group lung sections were prepared from three control IgG treated mice (+IAV) and three S100A9 Ab treated mice (+IAV). The lung sections were used for TUNEL staining. Image J software was used to calculate TUNEL-positive areas (representing apoptosis) in the lung sections as detailed in the methods section. The data is presented as percent apoptotic area. The percent apoptotic area was calculated from nine areas/lung section as detailed in the methods section. The values were compiled to calculate the percent apoptotic area in IAV infected IgG treated mice vs. IAV infected S100A9 Ab treated mice, * p = 0.0164 by Student's t test. ( B ) A representative TUNEL staining of lung sections from IAV infected mice administered with either IgG or S100A9 Ab. The apoptotic nuclei (representing apoptosis) are indicated with red arrows. ( C ) A schematic model depicting the role of extracellular S100A9 and DDX21/TRIF/S100A9/TLR4/MyD88 signaling network in exaggerating lung disease during IAV infection. PM, plasma membrane; NM, nuclear membrane.

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, TUNEL Assay, Staining, Software