Characterization of programmed cell death pathways activated in mycobacterium tuberculosis-infected THP-1 cells and human monocyte-derived macrophages

Abstract

Mycobacterium tuberculosis (Mtb) primarily infects human lung macrophages, which serve as its major replication niche. Mtb can manipulate host macrophage cell death pathways to its advantage by inhibiting apoptosis and inducing necrotic cell death. However, the specific necrotic cell death pathway activated in human macrophages after Mtb infection remains unclear. Here, we used the THP-1 cell line and primary human monocyte-derived macrophages (hMDMs) to analyze multiple programmed cell death pathways during days 1–3 after Mtb infection. Regarding apoptosis, confocal microscopic analysis demonstrates that Mtb rarely induces apoptosis in THP-1 cells or hMDMs. Immunoblotting shows that Mtb induces significant CASP3 and GSDME activation in THP-1 cells, but not in hMDMs. We show that Mtb induces a significant increase in GSDMD cleavage, a hallmark of pyroptosis, in THP-1 cells but not in hMDMs. MLKL phosphorylation was not observed in THP-1 cells or hMDMs during Mtb infections, indicating an absence of necroptosis. No changes in ferroptosis markers such as GPX4 expression or lipid peroxidation levels were detected. Time-lapse live-cell imaging revealed no lysosomal membrane permeabilization prior to plasma membrane rupture (PMR). However, we observed DNA release from Mtb-infected THP-1 cells and hMDMs after PMR. The DNA released from THP-1 cells exhibits low levels of myeloperoxidase and histone H3 citrullination. High-resolution confocal imaging shows that Mtb is associated with the released DNA. We demonstrate that pyroptosis induction is dispensable for DNA release and cell death induction. In conclusion, our results reveal that Mtb-triggered cell death in hMDMs bypasses canonical cell death pathways, including apoptosis, pyroptosis, necroptosis, and ferroptosis. Instead, cell death in both THP-1 cells and hMDMs correlates with DNA release that shares partial features with METosis or NETosis.