Figure 5: IL-6 impairs intracellular killing of M. tuberculosis through altered LD homeostasis.

(a,b) Screening of the effect of cytokines signaling through JAK–STAT pathway on intracellular survival of mycobacteria in human macrophages. (a) Differentiated THP-1 cells were incubated with a luminescent strain of M. bovis BCG (BCG-lux) for 2 h, repeatedly washed, then treated with cytokines (20 ng ml⁻¹) for 24 h at 37 °C. Cells were then lysed and luminescence measured. Viable intracellular mycobacteria were increased by IL-6 (red) and IL-5, and reduced by granuloyte–macrophage colony-stimulating factor and Leptin (green). Significance comparisons are between indicated treatment and untreated control. (b) Comparison of effects of treatment with IL-6 before and after infection in THP-1 cells. (c) Primary human macrophages were incubated with Bleupan M. tuberculosis for 2 h, repeatedly washed and then treated with IL-6 (red, at concentrations shown) for 24 h at 37 °C before cells were lysed and plated to count CFUs. Significance comparisons are between indicated treatment and untreated control. (d) ATG2A-V5 overexpression reduces intracellular neutral lipids in THP-1 cells. Scale bars, 10 μm. (e) Treatment of primary human macrophages with IL-6 (80 ng ml⁻¹ for 24 h) causes a small reduction in ATG2A protein abundance. Quantification is from three independent experiments. Error bars represent s.e.m. (f,g) BCG-induced LD accumulation in enhanced by IL-6 treatment. Representative images (f) and fluorescence quantification (g) of intracellular LDs (red), in primary human macrophages 24 h after infection with GFP-labelled M. bovis BCG (green). LDs are increased following infection, further increased in the presence of IL-6 treatment (80 ng ml⁻¹; red) and significantly reduced by pretreatment with the DGAT1 inhibitor T863 (10 μM, 48 h). Scale bars, 10 μm (f). (h) The increase in intracellular viable M. bovis BCG in primary human macrophages treated with IL-6 (red; 80 ng ml⁻¹ for 24 h post infection) is blocked by pretreatment with the DGAT1 inhibitor T863 (as above). *P<0.05, **P<0.005 and ***P<0.001. Figures are representative of at least three independent experiments with at least three replicates per data point.