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. 2013 Feb 27;8(2):e57307. doi: 10.1371/journal.pone.0057307

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© 2013 Zimmermann et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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GF/IL17 transgenic animals and littermate controls between 2 and 3 month were injected twice with 50 µg LPS i.p. in 24 hours or treated with mock injections of PBS. (A) Quantitative rt-PCR revealed a strong upregulation of the expression of inflammatory cytokines Tnf, Il1b, and Ccl2 by LPS treatment whereas Il6 was not induced following endotoxemia. This effect was markedly pronounced in GF/IL17 transgenic animals (Tnf: p < 0,01; Il1b: p < 0,05). Furthermore Ccl2 expression was strikingly upregulated in some of the LPS treated GF/IL17 mice compared with LPS treated wild-type controls but due to the high interindividual variance not considered as significant. (B) Representative flow cytometry profiles from mock- or LPS-treated GF/IL17 and WT mice. LPS treatment induced a population of CD45^high/CD11b⁺ activated microglia in both WT and GF/IL17 mice. Chronic IL-17A stimulation strikingly augmented this effect, respectively. The numbers above the indicated gate show the mean percentages of FSC/SSC gated populations. (C) For the statistical analysis of infiltrating cell numbers a ratio between CD45^high/CD11b⁺ activated microglia and CD45^dim/CD11b⁺ resting microglia was calculated for each individual mouse.GF/IL17 transgenic animals exhibited a significantly elevated ratio of CD45^high/CD11b⁺ activated to CD45^dim/CD11b⁺ microglia after LPS treatment, respectively (p < 0,05). (D) Lectin immunohistochemistry revealed a pronounced accumulation of activated microglia (arrows) in the periventricular regions (asterisk: choroid plexus) after LPS treatment in GF/IL17 mice. (E) Intracellular staining of TNF-α after LPS treatment. Only CD45 positive microglia and monocytes/macrophages are stained by anti TNF-α antibody (left histogram) whereas GLAST positive astrocytes are negative for TNF-α (right histogram). In GF/IL17 transgenic mice (light gray) compared with wildtype mice (dark gray) CD45 positive microglia and monocytes/macrophages exhibit a stronger intracellular TNF-α staining after LPS treatment.