Uptake‐independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates

A, B

Percentage of macrophages surviving after interaction with an extracellular Mtb aggregate originating from the debris of dead macrophages (A) or from axenic culture (B). Each line represents an independent experimental replicate (n = 3 replicates with ≥ 100 cells per replicate).

C

Percentage of macrophages that die within the first 12 h after stable contact with an Mtb aggregate originating either from a dead macrophage (MΦ) or from an axenic culture. Each symbol represents the percentage of events for a single experimental replicate (n ≥ 100 events per replicate). Bars represent means and standard deviations; P‐value calculated using a t‐test ns, P‐values > 0.05.

D

Percentage of macrophages that die by the end of the experiment (by 60 h post‐infection). Macrophages in “contact” represents the fraction of cells that interact with an Mtb aggregate during the course of the experiment. “Bystander” (byst.) macrophages are in the same sample as the infected ones but they do not establish physical contact with an Mtb aggregate during the course of the experiment. “Uninfected” (uninf.) macrophages are not exposed to Mtb. Each symbol represents the percentage of events for a single experimental replicate (n ≥ 50 events per replicate). Bars represent means and standard deviations; P‐value calculated using a one‐way ANOVA test, ns, P‐values > 0.05.

E, F

(E) Example of a macrophage that interacts with an Mtb aggregate (00:00 h), fragments it (03:00 h), redistributes the bacteria in a “bullseye” pattern around the nucleus (12:00 h), and dies (16:00 h). (F) Example of a macrophage that stably interacts with an Mtb aggregate without fragmenting it (00:00–09:00 h) and ultimately dies (12:00 h). In (E and F), Draq7 staining of the nucleus (in blue) is used as a marker for cell death. Scale bars, 20 μm.

G, H

BMDMs infected with aggregates of Mtb were imaged by time‐lapse microscopy (every 30 min for up to 13.5 h) followed by fixation, immunostaining (nuclei stained with Hoechst, membrane staining with anti‐CD45 antibody), and imaging by confocal microscopy. White arrows indicate intracellular bacteria, cyan arrows indicate extracellular bacteria. All scale bars, 10 μM. (G.I, H.I) Time‐lapse microscopy image series of macrophages that interacts with Mtb aggregates and fragment (G) or do not fragment (H) them. (G.II, H.II) Max intensity projection of confocal microscopy images of the same macrophages are shown in panels I. (G.III, H.III) 3‐D reconstruction of the cells imaged in panels II, images are cropped in x or y in the position indicated by the white dotted lines in panels II to show the inside of the cell.

I–M

BMDMs infected with aggregates of Mtb were imaged by time‐lapse fluorescence microscopy followed by SEM. (I) Example of a macrophage showing the typical “bullseye” pattern of bacterial redistribution around the cell nucleus. Scale bar, 10 μm. (J) Correlative SEM image of (I). (K) Example of a macrophage interacting with an extracellular Mtb aggregate without complete fragmentation and redistribution of bacteria. White arrows indicate intracellular bacteria that have been detached from the main aggregate and internalized. Scale bar, 10 μm. Time‐lapse microscopy image series of these macrophages are shown in Appendix Fig S4A. (L, M) Correlative SEM image of (K). Scale bar, 2 μm (M).

N

Percentage of macrophage‐Mtb aggregate interactions resulting (“fragm.,” pattern exemplified by panel E) or not resulting in fragmentation (“not fragm.,” pattern exemplified by panel F) of the Mtb aggregate. Quantification was performed by manual annotation of all the macrophage‐Mtb aggregate interactions observed in the time‐lapse microscopy movies. Each symbol represents the percentage of events for a single biological replicate (n ≥ 100 events per replicate). Bars represent means and standard deviations.

O

Percentage of macrophages surviving over time after uptake and fragmentation of Mtb aggregates or after contact without fragmentation. Each line represents an independent biological replicate. The vertical dotted line marks the 12 h post‐interaction time‐point.

P, Q

BMDMs were treated with cytochalasin D to prevent bacterial uptake, infected with aggregates of Mtb, and imaged by time‐lapse microscopy at 1‐h intervals for 60 h. (P) Percentage survival over time for macrophages in contact with Mtb aggregates. Time 0 represents the time when stable contact with an aggregate begins. Each line represents an independent biological replicate (n = 3 replicates with ≥ 100 cells per replicate). (Q) Percentage of cytochalasin D‐treated macrophages that die by the end of the experiment (by 60 h post‐infection). Each symbol represents the percentage of events for a single biological replicate (n ≥ 100 events per replicate). Bars represent means and standard deviations; P‐value calculated using a one‐way ANOVA test; ns, P‐values > 0.05.

Figure 1. Aggregates of Mtb kill macrophages in a contact‐dependent but uptake‐independent manner.