Figure 2. Mst1 and Mst2 deficient myeloid cells are defective in bacterial killing and ROS induction.
(a) Mst1fl/flMst2fl/fl (WT) and Mst1fl/flMst2fl/flLyz2-Cre (cDKO) BMDMs left uninfected or infected for 10 min at 37°C or 4°C with FITC-labelled E. coli (E. coli-FITC) at a multiplicity of infection (MOI) of 20 or L. monocytogenes (Lm-FITC) at MOI of 10, as analysed by flow cytometry.
(b) WT and cDKO BMDMs or neutrophils were infected with L. monocytogenes or E. coli (MOI, 10). CFUs were quantified at the indicated time points.
(c, d) WT and cDKO BMDMs infected with GFP-expressing E. coli (GFP-E. coli) for the indicated times were washed, fixed and stained with DAPI. The GFP-E. coli (green) were visualized (c) and quantified (d) by fluorescence microscopy. Scale bar, 20 μm.
(e) WT and cDKO BMDMs or neutrophils were stained with MitoSOX (mROS) or CM-H2DCFDA (cellular ROS) for 30 min and analysed by flow cytometry.
(f) WT (top panels) or cDKO BMDMs (lower panels) were labeled with CFSE, and then mixed with unlabeled cDKO or WT cells and the CellROX and DAPI coupled E. coli. The phagosomal ROS was visualized by fluorescence microscopy. WT and cDKO cells indicated with stars and arrows respectively. Scale bar, 20 μm.
(g) WT and cDKO BMDMs or neutrophils were stimulated with the indicated TLR agonists for 6 h, and mROS production was measured with MitoSOX using flow cytometry. LPS, Lipopolysaccharide; Pam3, Pam3CSK4; LTA, Lipoteichoic acid.
(h) mROS and cellular ROS in WT and cDKO BMDMs or neutrophils were measured as described in (e) after E. coli or L. monocytogenes infection. Data were assessed with Student's t test and are represented as mean ± s.d. n=5, biological replicates. * p<0.05, ** p<0.01, *** p<0.001 compared with respective controls or as indicated (b, d). Data are from one experiment representative of two (f) or three (a-e, g, h) independent experiments with similar results.