Runx proteins mediate protective immunity against Leishmania donovani infection by promoting CD40 expression on dendritic cells
Fig 5
LD attenuates LPS- and TNFα-induced Runx binding to the CD40 promoter by inhibiting PI3K-Akt pathway.
(A) EMSA (probes as in Fig 2) evaluating the binding of Runx1 and Runx3 to the CD40 promoter (top), and immunoblot analysis showing Runx1 and Runx3 expression (bottom) in BMDCs left uninfected (Uninf) or infected with LDPm for indicated times and then stimulated with (+) LPS (left panels) or TNFα (right panels) for 0.5 h or left unstimulated (-). Numbers below lanes represent densitometry (as in Fig 4B); presented relative to uninfected BMDCs that had been left unstimulated. A compilation of densitometry results from three independent experiments is shown in S11 Fig. (B) Flow cytometry analysis of TLR4, TNFR1 and TNFR2 expression on BMDCs left uninfected or infected with LDPm for indicated times. (C) Confocal microscopy of translocation of Runx1 or Runx3 (red) into the nuclei (blue; Hoechst staining) in BMDCs left uninfected or infected with LDPm for 24 h and then stimulated with LPS or TNFα for 0.5 h or left unstimulated (US). Pink color (merge) shows nuclear translocation of Runx1 or Runx3. Scale bar, 10 μm. (D and E) Nuclear lysates were prepared from BMDCs left uninfected or infected with LDAm for 24 h and then kept untreated (-) or treated (+) for 0.5 h with LPS or TNFα (D). In some experiments (E), nuclear lysates were prepared from sDCs that were derived from uninfected (Uninf mice) or LD-infected mice (Inf mice; isolated on day 45 postinfection) and then treated with LPS for 0.5 h. Lysates were subjected to EMSA (probes as in Fig 2) to detect the binding of Runx1 and Runx3 to the CD40 promoter. Numbers below lanes indicate relative densitometry quantification of Runx1 and Runx3 binding to the CD40 promoter (normalized to OCT-1 binding). (F and G) Immunoblot analysis of total and phosphorylated Akt in lysates of BMDCs infected with LDPm (F) or LDAm (G) for 24 h or left uninfected and then treated with LPS (left panels) or TNFα (right panels) for 0.3 h. Below lanes, densitometry, normalized to total Akt and presented relative to uninfected BMDCs given control treatment (neither LPS nor TNFα). In all experiments, LD strain AG83 was used. Data are representative of three (A, B, and D-G) or two (C) independent experiments.