Fig. 2

The protective effect of dexamethasone is mediated by MKP-1 and p38 MAPK inhibition. a Inhibition of MKP-1 by (E/Z)-Bcl hydrochloride or inhibition of the glucocorticoid receptor (GR) by Ru-486 in Mtb-infected MRC-5 lung fibroblasts (MOI 10) abrogates the protective effect of dexamethasone (5 µM) on host cell survival. Viable fibroblasts were quantified 72 h post infection using PrestoBlue. b Protective effect of the GR agonist BI653048 (10 µM) in Mtb-infected human Mφ (MOI 1). Cells were stained with DAPI and the number of living cells was determined 48 h post infection. c Dexamethasone (5 µM) treatment increases MKP-1 expression in infected MRC-5 lung fibroblasts 5 h after infection. Relative expression of MKP-1 was measured by quantitative real-time PCR (qRT-PCR). d–f Mtb induces p38 MAPK activation in J774.2 Mφ (d, e) and MRC-5 lung fibroblasts (f). Whole-cell lysates were obtained from infected cells treated with or without dexamethasone (5 µM) and equal amounts of protein were subjected to western blot analysis to determine the levels of phosphorylated and total p38 MAPK. β-Actin was used as a loading control. Images are representative of two to three individual experiments. g, h Cytoprotective effect of the p38 MAPK inhibitor doramapimod (10 µM) in Mtb-infected human Mφ from healthy donors (g) and TB patients (h). Cells were stained with DAPI and the number of living cells was determined 48 h post infection. i Doramapimod (10 µM) inhibits Mtb-induced p38 MAPK phosphorylation. Quantification of phosphorylated p38 MAPK in MRC-5 lung fibroblasts infected with Mtb and treated with the p38 MAPK inhibitors BMS-582949 (10 µM) or doramapimod by western blot. j Knock-down of p38 MAPK in Mtb-infected J774A.1 Mφ reduces mycobacterial cytotoxicity. Infected cells (MOI 3 and 5) were stained with DAPI and the number of surviving cells was determined after 48 h of infection. Data from one experiment with multiple replicates (a, b) or data pooled from four individual experiments (c) are shown; data from two individual experiments are shown in e and h; data from three independent experiments (g, j) with multiple replicates are shown as mean ± SEM. Analysis was done using unpaired t- test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)