Figure 1.
Impaired intracellular control of BCG growth by PuM upon IFN-γ treatment.
BMM and PuM were infected with GFP-BCG. After infection, cells were left untreated or treated with IFN-γ (20, 40 and 80 ng/ml) for 48 h. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of a representative experiment from 3 independent experiments with 4 replicates each. The differences between groups of BMM and PuM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * significantly different from medium control, P<0.05.
Figure 2.
PuM are specifically unresponsive to IFN-γ in the control of intracellular bacteria.
PuM, AM and PEM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup for 48 h or left untreated. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of 2 independent experiments with 4 replicates each. The differences between groups of PuM, AM and PEM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * significantly different from medium control, P<0.05.
Figure 3.
IFN-γ and AECsup induce different cytokine profiles in PuM and BMM.
BMM and PuM were infected with GFP-BCG. After infection, cells were left untreated or treated with either IFN-γ or AECsup. Cell culture supernatants were collected at 4 and 24 h after treatments. IL-12, IP-10 and IL-6, levels were measured using ELISA. Values are expressed as means ± SD, from 3 independent experiments. The differences between groups of PuM and BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test, * Significantly different from medium control, p<0.05.
Figure 4.
Impaired intracellular control of BCG growth by PuM upon IFN-γ treatment is regulated by SOCS1.
PuM from IFN-γ-/- SOCS1-/- mice were infected with GFP-BCG. After infection, cells were left untreated or treated with IFN-γ, AECsup and IFN-γ + AECsup for 48 h. Bacterial growth was evaluated by determining RLU. Data are shown as % reduction of phagocytosed bacteria evaluated as RLU. Values are means ± SD of the mean of a representative experiment from 2 independent experiments with 4 replicates. The differences between groups of IFN-γ-/- SOCS1-/- PuM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P<0.05.
Figure 5.
AECsup has opposite effects to IFN-γ on iNOS, and Arg-1 expression in BMM and PuM and mediates killing through an iNOS independent mechanism.
BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup or 1 mM of NG-monomethyl-L-arginine (NMMLA) together with either IFN-γ or AECsup or left untreated. Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of a) iNOS, b) Arg-1 ± SD from 3-4 experiments. In c) the effect of the iNOS inhibitor NMMLA on intracellular growth of BCG is shown. Bacterial growth was evaluated by determining RLU. Data are expressed as mean ± SD. The differences between groups of BMM and PuM were analyzed using non-parametric, one-way ANOVA (Kruskal-Wallis) with Dunn‘s post-test. * significantly different from medium control, P<0.05
Figure 6.
AECsup induces IL-1β expression and secretion without an effect of intracellular killing of BCG.
BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup or left untreated. a) Total RNA was extracted from both cell-types after 4 and 24 h of treatment and the mean-fold accumulation of IL-1β transcripts shown. Data shows the average of 3 independent experiments. b) Cell culture supernatants were collected 24 h after infection and IL-1β measured with ELISA. Data shows the average of 6 independent experiments. c) The effect of incubating cells with IL-1β (1000 pg/ml) on intracellular growth of BCG. Data representative of 3 independent experiments d) The effect of AECsup on intracellular growth of BCG in BMM from caspase1-/- mice. Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. The differences between groups of BMM were analyzed using a one-way ANOVA followed by Bonferroni‘s Multiple Comparison Test. * significantly different from medium control, P<0.05.
Figure 7.
IFN-γ and AECsup induce an oxidative burst following infection with BCG.
BMM were treated with IFN-γ (20 ng/ml) or AECsup 24 h prior to infection and then infected with GFP-BCG. a) ROS production was monitored in the cultures for 16 h by measuring luminescence with luminol as substrate. b) The effect of incubating cells with superoxide dismutase (SOD) and catalase on intracellular killing of BCG is shown. Values are expressed as means ± SD from 5 wells. Differences between treatments with or without SOD/catalase were analyzed using a paired t-test. * significant effect of SOD/catalase treatment, P<0.05.
Figure 8.
Inhibiting phagosomal acidification does not affect intracellular killing of BCG.
BMM and PuM were infected with GFP-BCG. After infection, cells were treated with either IFN-γ or AECsup with or without chloroquine (10 µM). Bacterial growth was evaluated by determining RLU. Values are expressed as means ± SD from 5 wells. The data is representative of 2 independent experiments. Differences between treatments analyzed using a one-way ANOVA followed by Bonferroni's Multiple Comparison Test. * significantly different from medium control, P<0.05.