Fig. 4

Mtb induces RIPK1 independent necrotic host cell death. a, b Mtb-infected MRC-5 lung fibroblasts were treated with doramapimod (10 µM). After 72 h lactate dehydrogenase (LDH) was quantified in the culture medium of infected cells (a). Mtb-infected human Mφ derived from TB patients were treated with doramapimod and LDH release was measured 48 h post infection in the supernatant (b). Data are representative of two individual experiments. c Dexamethasone (5 µM) and doramapimod (10 µM) reduce the release of the necrosis marker High-Mobility Group Protein B1 (HMGB1) in Mtb-infected MRC-5 lung fibroblasts. d Protective effect of necrostatin (Nec; 10 µM), etanercept (10 µg/ml), adalimumab (50 µg/ml), and thalidomide (10 µM) in Mtb-infected human Mφ from healthy donors. Cell viability was quantified by DAPI staining 48 h after infection. e Bone-marrow-derived Mφ (BMDM) from WT and mixed lineage kinase domain-like pseudokinase (MLKL)^−/− mice were infected with Mtb (MOI 10) and cell survival was assessed 48 h after infection using DAPI staining. f BMDM from WT and tumor necrosis factor receptor (TNFR)^−/− mice was infected with Mtb (MOI 10). Cell survival was determined 48 h after infection using DAPI staining. g MRC-5 lung fibroblasts were treated with Z-VAD-FMK (10 µM) and necrostatin-1 (10 µM) simultaneously and viable cells were analyzed 72 h post infection with TB using PrestoBlue. Representative data from at least two experiments with multiple replicates are shown in a, b, d–f. And data pooled from eight independent experiments are shown in c. Representative data from two experiments with multiple replicates are shown in g. Results are expressed as mean ± SEM. Analysis was done using unpaired t-test (ns not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)