Fig. 4

a-c Increase of A. phagocytophilum 16S rRNA relative to murine HPRT mRNA at different time points p.i. of wild-type (WT) (a), IFN-γ R^−/− (b) and iNOS^−/− (c) neutrophils. Half of the set of samples was stimulated with IFN-γ. Results were normalised to the respective 0 h value of each sample using the ΔΔC_t-method. Mean and SD from 5 independent experiments are shown. Differences between unstimulated and IFN-γ-stimulated groups at each time point were analysed using the two-tailed Mann-Whitney test. *P < 0.05, **P < 0.01. d, f Relative iNOS mRNA expression normalised to murine HPRT at different time points in uninfected, IFN-γ-stimulated, A. phagocytophilum-infected, A. phagocytophilum-infected + INF-γ-stimulated, and LPS-stimulated (200 ng/ml) WT (d) and IFN-γ R^−/− (f) Hoxb8 neutrophils. Results were normalised to the 0 h value of uninfected WT cells using the ΔΔC_t-method. Mean and SD from 5 independent experiments are shown. Differences between experimental groups were analysed using the two-tailed Mann-Whitney test. Stimulated and/or infected WT and IFN-γ R^−/− Hoxb8 neutrophils were compared to the respective uninfected controls. *P < 0.05, **P < 0.01. Further, a significant difference was detected between infected and infected + IFN-γ-stimulated WT cells at time points 24 h, 48 h, 72 h (P < 0.01) and 92 h (P < 0.05) p.i. The same was not true for IFN-γ R^−/− Hoxb8 neutrophils. e Nitrite accumulation as a marker for iNOS activity was measured in the supernatants of uninfected (medium), A. phagocytophilum-infected, IFN-γ-stimulated, A. phagocytophilum-infected + IFN-γ-stimulated, and LPS-stimulated (200 ng/ml) WT Hoxb8 neutrophils by Griess assay. Mean and SD from 5 independent experiments are shown. Differences between infected and/or stimulated cells and the medium controls at each time point were analysed using the two-tailed Mann-Whitney test. **P < 0.01