Fig 1: Neutralization of IFNγ and TNF alter the inflammatory environment induced by secondary challenge. Naïve and LCMV-immune mice were challenged with Lm-gp61 and some groups of mice were additionally treated with neutralizing antibodies to IFNγ or TNF. Bar plots show the concentration of (A) IFNγ; (B) TNF and (C) IL-6 in the serum of the indicated treatment groups at day 1 or 3 after Lm-gp61 challenge. n = 7–8 mice per group, data are representative of two separate experiments.
Fig 2: Neutralization of TNF prevents accumulation of activated macrophages in the spleen. Naïve and LCMV-immune mice were challenged with Lm-gp61 and some groups of mice were additionally treated with neutralizing antibodies to IFNγ or TNF. (A) Splenocytes were gated for F4/80+CD11b+ macrophages. The bar graph indicates the frequency of macrophages expressing IFNγR1 (CD119) for each treatment group. Accompanying flow plots indicate representative gating for CD119+ macrophages, as compared to isotype control. Frequencies were obtained by subtracting the percentage that were stained by the isotype control; (B) The bar graph indicates the change in MFI of CD119 staining on F4/80+CD11b+ macrophages for each treatment group. The change in MFI was obtained by subtracting the MFI following isotype control staining from the MFI following CD119 staining. The accompanying flow plot indicates representative staining for CD119 on macrophages for each treatment group; (C) RNA from FACS-sorted F4/80+CD11b+ macrophages was analyzed by semi-quantitative RT-PCR for changes in cytokine transcript levels. Bar plots indicate the relative fold change in expression between treatment groups for the indicated transcripts. n = 3–5 mice per group.
Fig 3: Heterologous challenge with Lm-gp61 induces increased levels of serum IFNγ. Naïve and LCMV-immune mice were challenged with Lm-gp61. Cytokine concentrations in the serum at day 1 and 3 post-infection were assessed using a cytokine bead array for (A) IFNγ; (B) IL-12p70; (C) TNF; (D) IL-1β; (E) IL-10; and (F) IL-6. n = 8 mice/group, data are pooled from two separate experiments. Dotted lines indicate limit of detection for the assay.
Fig 4: Protection mediated by CD4+ memory T cells is heavily dependent on TNF but only partly dependent on IFNγ. Naïve and LCMV-immune mice were challenged with Lm-gp61 and some groups of mice were additionally treated with neutralizing antibodies to IFNγ or TNF. Bar graphs indicate bacterial load in the (A) spleen and (B) liver day 3 following primary or secondary challenge (n = 4–7 mice/group); (C,D) Bar graphs indicate bacterial load in the spleen and liver, as indicated, on day 3 following primary Lm-gp61 infection with or without TNF neutralization (n = 4 mice/group); (E) Bar graph shows bacterial burden in the spleen of LCMV-immune LysMCre/Ifngr1fl/fl mice day 3 after rechallenge with Lm-gp61 (n = 4 mice/group). Data are representative of at least two separate experiments.
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