A – B. Dendritic cells (DCs) were infected, at MOI 10 with live/dead H37Ra or live/dead H37Rv. (U = uninfected, LH37Ra = live H37Ra, sH37Ra = streptomycin-killed H37Ra, LH37Rv = live H37Rv, iH37Rv = γ-irradiated H37Rv.) Cell death was measured by propidium iodide exclusion (A) 72 h post-infection or (B) 24 h post-infection on a GE IN Cell

Analyzer 1000. (A – B) are means (± SEM) Vemurafenib datasheet of 3 pooled donors. * p < 0.05 vs. Uninfected. C. DCs were infected with live H37Ra at MOI 1, 5 or 10. Cell death was measured by propidium iodide exclusion 72 h after infection. Staurosporine was used as a positive control for cell death. * p < 0.05 vs. uninfected, ns - not significantly different from uninfected. D. DCs were infected with live H37Ra at MOI 1, 5 or 10. DNA fragmentation was measured by Cell Death ELISA 72 h after infection. * p < 0.05 vs. Uninfected, ns - not significantly different from uninfected. E. DCs were infected with live H37Ra at MOI 10 for 72 h. Nuclei were stained with Hoechst and visualised by fluorescence microscopy. Cycloheximide and staurosporine were used as positive controls for nuclear fragmentation. (C - E) are 1 representative donor of 3, showing means (± SEM) of 3 independent wells. Having established that reduced DC viability was dependent on

infection with live mycobacteria, we then investigated the mechanism of cell death in H37Ra-infected DCs. We previously noted that macrophage cell death after Palbociclib Mtb infection results in DNA fragmentation. By ELISA, we could show that DNA fragmentation

was also a feature of the DC PAK5 response to viable Mtb H37Ra infection peaking at an MOI of 5 (Figure 2D). Apoptosis results in nuclear condensation, pyknosis and, eventually, fragmentation of the nucleus into apoptotic bodies [20, 21]. To determine whether this occurred during Mtb H37Ra infection, the nuclear morphology of DCs stained with Hoechst was examined by epifluorescent microscopy. The nuclei of infected cells did not undergo pyknosis or fragmentation and were similar in appearance to those of uninfected cells at 72 h after infection, a time at which they had undergone significant cell death. DCs treated with cycloheximide and staurosporine displayed extensive nuclear fragmentation, indicating that the cells are capable of undergoing this process when treated with apoptotic stimuli (Figure 2E). Dendritic cell death after M. tuberculosis H37Ra infection is caspase-independent and proceeds without the activation of caspase 3 and 7 Activation of caspases is considered to be essential for classical apoptosis [22]. Therefore, we sought to establish if DC death following Mtb infection was caspase dependent. Cells were treated with the pan-caspase inhibitor Q-VD-OPh and infected with H37Ra, at an MOI of 10, and cell death was assessed using IN Cell fluorescent microscopy and analysed as before.