Figure 3.

Phagocytosis of Mtb is crucial for the induction of Irg1 gene expression. (A–C) Macrophages were pretreated with the phagocytosis inhibitors Mycalolide B (MycB; 1 µM, 3 µM or 6 µM) or Dynasore (10 µM, 50 µM or 100 µM) for 1 h and then infected with H37Rv-RFP or stimulated with LPS (10 ng/ml) as indicated. (A) Phagocytosis was determined by flow cytometric analysis at 6 h after H37Rv-RFP Mtb exposure (MOI of 1). FACS plot (left panel) and summary data (right planel) are shown. (B) Irg1 expression was evaluated in BL/6 BMDMs exposed to latex beads (0.025%) (upper graph) or to H37Rv treated or not with MycB and Dynasore as well as in cultures left at 4 ⁰C. (C) TLR-2 protein expression by Mtb-infected macrophages following MycB and Dynasore treatment assessed by flow cytometry. (D) Irg1 mRNA levels were determined in BMDMs exposed to H37Rv opsonized or not with fresh or heat-inactivated naïve mouse sera at 3 h and 6 h p.i. Statistically significant differences are indicated as follows: **p < 0.01 and ***p < 0.001. Data are representative of two separate experiments.