Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

Abstract

BackgroundDendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival.ResultsInfection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth.ConclusionsHuman DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

Cigarette Smoking Impairs Human Pulmonary Immunity to Mycobacterium tuberculosis

“…Cell viability was assessed using the propidium iodide (PI) exclusion method for plasma membrane integrity of cells, and the nuclei were counterstained with Hoechst as previously described by us (24,26). The number of PI-positive cells relative to the total number of nuclei per field was counted by automated fluorescence microscopy using the IN Cell Analyzer 1000 and IN Cell Investigator software (GE Healthcare, Pittsburgh, PA).…”

The interplay between pathogen‐associated and danger‐associated molecular patterns: an inflammatory code in cancer?

“…(4) PAMPs can induce the release of DAMPs: thus infection processes, such as infection of airway epithelial cells by respiratory syncytial virus, induce the release of hsp27, activating neutrophils via TLR4, 43 and DCs infected with M. tuberculosis undergo caspase‐independent cell death that allows the release of inflammatory DAMPs, not impeded by caspase neutralization 44 …”

“…(4) PAMPs can induce the release of DAMPs: thus infection processes, such as infection of airway epithelial cells by respiratory syncytial virus, induce the release of hsp27, activating neutrophils via TLR4, 43 and DCs infected with M. tuberculosis undergo caspaseindependent cell death that allows the release of inflammatory DAMPs, not impeded by caspase neutralization. 44 Owing to similarities to IL-1a and HMGB1 in regard to constitutive nuclear expression and passive release, it has been suggested that IL-33 is a member of the alarmin (or DAMPs) family, 45 and the active release of IL-33 by macrophages can be triggered by PAMPs, such as LPS. 46 Taking all this into account, we assemble a tentative PAMP-DAMPbased inflammatory code.…”

Networked T Cell Death Following Macrophage Infection By Mycobacterium Tuberculosis

“…Following 24 h incubation, cells were stained simultaneously with 5 mg/ml Propidium Iodide (PI), 20 mg/ml Hoechst 33342 and 50 mg/ml Hoechst 33258, briefly resuspended by gentle pipetting, then plates were centrifuged at 4006g for 2 min to bring cells in suspension to the bottom surface of the well. Plates were then imaged using an INCell Analyzer 1000 cellular imaging system (GE Healthcare) and images were analyzed using GE INCell Analyzer 1000 Workstation software version 3.6 (GE Healthcare) as we have previously published [21]. Total cell numbers were detected via Hoechst staining of nuclei, and the dying/dead cells were identified via positivity for PI staining and/ or nuclear condensation, which was characterised by elevated intensity of Hoechst staining within the nuclei (see figure S1).…”