Journal: The EMBO Journal

Article Title: Loss of T‐bet confers survival advantage to influenza–bacterial superinfection

doi: 10.15252/embj.201899176

Figure Lengend Snippet: A Wild‐type mice were infected with indicated doses of S. pneumoniae serotype 19F or S3 bacteria alone and assessed for survival. B–F Wild‐type and T‐bet −/− mice were infected with sub‐lethal dose of influenza virus followed by sub‐lethal dose of secondary bacterial challenge ( S. pneumoniae serotype S3, 50 cfu) 7 days later. (B) Survival of mice following post‐influenza bacterial infection. (C) Pneumococcal cfu in lung homogenates of mice infected with 50 cfu of S. pneumoniae serotype S3 alone or following influenza virus infection. (D) Absolute immune cell counts in lungs, as assessed by flow cytometry. (E) Concentrations of IL‐17 and IL‐22 in lung homogenates of mice, assessed by ELISA. (F) T‐bet −/− mice were administered αIL‐17 or IgG isotype control antibody every other day from day −2 post‐secondary bacterial infection and monitored for survival. Data information: Data in (A–C) and (F) are combined from two independent experiments with 6–16 mice per group. Data in (D) and (E) are representative of two independent experiments with four mice per group. All data are represented as mean ± SEM. Statistical significance was determined by log‐rank test in (B) and (F), Mann–Whitney U ‐test in (C) and unpaired Student's t ‐test in (D) and (E).

Article Snippet: Cytokine neutralization was performed by administering a total of 100 μg of anti‐IL‐17A monoclonal antibody (BioXCell, Clone 17F3), 250 μg of anti‐IL‐22 monoclonal antibody (Genentech, Clone 8E11) or equal amounts of mouse IgG1 isotype control antibody (Genentech) per mouse on each dose.

Techniques: Infection, Bacteria, Virus, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Loss of T‐bet confers survival advantage to influenza–bacterial superinfection

doi: 10.15252/embj.201899176

Figure Lengend Snippet: Mice were superinfected as described in Fig A. Bacterial load as determined by enumeration of cfu in BAL following administration of αIL‐17 or αIL‐22 blocking antibody. Control wild‐type and T‐bet −/− groups were administered with IgG isotype control antibody. T‐bet −/− mice were administered αIL‐17 or IgG isotype control antibody every other day from day −2 post‐secondary bacterial infection and monitored for survival. Absolute neutrophil counts in the lungs following administration of αIL‐17 or IgG isotype control antibody, as determined by flow cytometry. Pneumococcal cfu in BAL following αLy6G antibody‐mediated neutrophil depletion. Control T‐bet −/− mice were administered with IgG isotype control antibody. See also Fig A. mRNA expression of antimicrobial peptides in lungs following administration of αIL‐17, IL‐22 or IgG control antibody, as determined by qPCR. Data information: Data in (A), (B) and (D) are combined from two independent experiments ( n = 8–12 mice per group). Data in (C) and (E) are representative of two independent experiments with 3–4 in (C) or 4–6 in (E) mice per group. All data are represented as mean ± SEM. Statistical significance was determined with Kruskal–Wallis test followed by Dunn's multiple comparisons test in (A), log‐rank test in (B), unpaired Student's t ‐test in (C), Mann–Whitney U ‐test in (D) and one‐way ANOVA followed by Tukey's multiple comparisons test in (E). n.s., not significant. See also Fig B and C.

Article Snippet: Cytokine neutralization was performed by administering a total of 100 μg of anti‐IL‐17A monoclonal antibody (BioXCell, Clone 17F3), 250 μg of anti‐IL‐22 monoclonal antibody (Genentech, Clone 8E11) or equal amounts of mouse IgG1 isotype control antibody (Genentech) per mouse on each dose.

Techniques: Blocking Assay, Control, Infection, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Loss of T‐bet confers survival advantage to influenza–bacterial superinfection

doi: 10.15252/embj.201899176

Figure Lengend Snippet: Mice were infected as described in Fig A or infected with sub‐lethal influenza virus alone (B, left panel). Frequency and absolute numbers of pulmonary neutrophil depletion in T‐bet −/− lungs following αLy6G‐mediated depletion compared to IgG‐administered controls as assessed by flow cytometry. Numbers in flow cytometry plots indicate frequency of gated cells in each throughput. Relative amounts of PR8 viral NP mRNA following αIL‐17 treatment from day 0 (virus only infection, left panel) or from day 5 (bacterial superinfection, right panel) post‐influenza virus infection. Subsequent doses were administered every other day. Wild‐type and control T‐bet −/− mice were given IgG isotype control antibody treatment. Relative amount of PR8 viral NP mRNA following αLy6G or IgG isotype control antibody treatment as assessed by qPCR. Data information: Data are representative of three (A) or two (B and C) independent experiments with four mice per group and represented as mean ± SEM. Statistical significance in (A) and (C) was determined with unpaired Student's t ‐test.

Article Snippet: Cytokine neutralization was performed by administering a total of 100 μg of anti‐IL‐17A monoclonal antibody (BioXCell, Clone 17F3), 250 μg of anti‐IL‐22 monoclonal antibody (Genentech, Clone 8E11) or equal amounts of mouse IgG1 isotype control antibody (Genentech) per mouse on each dose.

Techniques: Infection, Virus, Flow Cytometry, Control

Journal: The Journal of Experimental Medicine

Article Title: Acetate coordinates neutrophil and ILC3 responses against C. difficile through FFAR2

doi: 10.1084/jem.20190489

Figure Lengend Snippet: IL-22–producing ILC3s are essential for acetate-mediated protection during CDI. (A–D) Clinical score (A), body weight changes (B), colon length (C), and intestinal permeability (D) in ILC3-sufficient ( AhR fl/fl ) and -deficient ( AhR fl/fl × Rorc-Cre ) mice that were treated (Ac) or not (Ct) with acetate and infected with C. difficile ( n = 5–8). AhR fl/fl × Rorc-Cre and AhR fl/fl littermates were infected at the same time. (E and F) Bacterial translocation into the liver (E) and spleen (F) in ILC3-sufficient and -deficient mice treated or not with acetate and infected with C. difficile . Samples were collected 5 d p.i., plated in blood agar, and incubated for 4 d at 37°C in aerobic or anaerobic conditions ( n = 5–8). (G) Infected mice were treated with or without acetate and received an i.p. dose of anti–IL-22 neutralizing antibody or isotype (iso) control IgG2a on days 1 and 3 p.i. (H and I) Mice were monitored for clinical score (H) and weight change (I) until day 5 p.i. ( n = 5). (J and K) Clinical score (J) and weight changes (K) of IL22 −/− and WT mice infected with 10 8 CFU of C. difficile that were either treated or not with acetate ( n = 5–6). IL22 −/− and WT mice were bred in the same animal facility, matched for sex and age, and infected at the same time to avoid batch effects. (L) Bacterial translocation into peripheral organs on day 2 p.i. in IL22 −/− mice treated or not with acetate ( n = 5–6). (M) Intestinal permeability of IL22 −/− mice treated or not with acetate and infected with C. difficile ( n = 5–6). Infected WT mice not treated with acetate are shown as controls. Results are representative of at least two independent experiments with four to six mice in each experimental group (G–I) or pooled results from two independent experiments with three to four mice in each group (A–F). Results are presented as mean ± SEM. *, P < 0.05.

Article Snippet: C. difficile –infected mice, treated or not with acetate in the drinking water, received an i.p. dose of 50 μl PBS with 150 g anti-mouse IL-22 neutralizing antibody (clone 8E11; Genentech) or an equivalent amount of isotype control IgG2a (BioXcell) on days 1 and 3 p.i.

Techniques: Permeability, Infection, Translocation Assay, Incubation, Control

Journal: The Journal of Experimental Medicine

Article Title: Acetate coordinates neutrophil and ILC3 responses against C. difficile through FFAR2

doi: 10.1084/jem.20190489

Figure Lengend Snippet: Reagents

Article Snippet: C. difficile –infected mice, treated or not with acetate in the drinking water, received an i.p. dose of 50 μl PBS with 150 g anti-mouse IL-22 neutralizing antibody (clone 8E11; Genentech) or an equivalent amount of isotype control IgG2a (BioXcell) on days 1 and 3 p.i.

Techniques: Control, Recombinant, Giemsa Stain, Modification, Staining, SYBR Green Assay, DNA Purification, Software, Real-time Polymerase Chain Reaction