Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages

Abstract

Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-α, IL-1β and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.

Fig. 1

Mtb induces NET formation in neutrophils. a Neutrophils were activated with Mtb (MOI 10), PMA (25 nM) or left inactivated and incubated for 3 h. Mtb-activated neutrophils acquire more clustered NETs than neutrophils chemically induced with PMA. b Quantitation of the DNA area in neutrophils stained with cell-impermeable Sytox Green with a frequency graph showing the percentage of Sytox-positive cells against the distribution of the DNA area. Mtb activation leads to more neutrophils with a larger DNA area compared to PMA activation and unstimulated neutrophils. c Bar graph showing the percentage of netting neutrophils with a DNA area >400 µm² as quantified from b. Mean values or representative micrographs from duplicates from 3 independent experiments are shown. * p < 0.05 and ** p < 0.01, compared to unstimulated control.

Fig. 2

Time kinetics of NET formation in neutrophils. a NETs were quantitated measuring fluorescence in neutrophils stained with cell-impermeable Sytox Green and activated with Mtb (MOI 10), PMA (25 nM) or left inactivated. The ratio of NETotic cells compared to a lysis control was followed for 18 h. b-d Effect of DPI or NEi on NET formation in untreated (b), Mtb-treated (c) and PMA-treated neutrophils (d). Mean values and error bars representing SEM from triplicates from 3 separate experiments are shown. * p < 0.05; ** p < 0.01.

Fig. 3

Mtb-induced NETs are phagocytosis dependent but not dependent on the bacterial 19-kDa lipoprotein. a Neutrophils exposed to wild-type Mtb (MOI 10) for 6 h. b, c Neutrophils treated with 10 µM CytD and activated with wild-type Mtb (b), or left inactivated (c). d Neutrophils exposed to an Mtb lspA−/− mutant lacking the 19-kDa bacterial cell wall lipoproteins. Representative micrographs from duplicates from 3 independent experiments are shown.

Fig. 4

Macrophages interact with NETs. Neutrophils were activated with Mtb (MOI 10) and incubated for 6 h. Macrophages (green) prestained with CFMDA were added to the wells, and coincubation was allowed for 1 h before cells were fixed. DNA was visualized with DAPI (blue), and neutrophil elastase was detected by antibody staining (red). a, b Arrows point to events of macrophage binding to elastase in NETs. c Arrow points to phagocytosed neutrophil elastase in macrophages.

Fig. 5

a Macrophages release increased amounts of cytokines in response to Mtb-activated neutrophils. b Effects of DPI and NEi on cytokine release. Neutrophils were preincubated for 30 min with DPI or NEi and activated with Mtb (MOI 10), PMA (25 nM) or left inactivated for 4 h at which macrophages were added to the wells and cells coincubated for 18 h. Mean values and error bars representing SEM from duplicates from 8 separate experiments are shown.* p < 0.05; ** p < 0.01.

Fig. 6

Hsp72 release in NET-forming neutrophils. a Mtb-activated neutrophils. b Mtb-activated neutrophils with DPI. c PMA-induced neutrophils. d Unstimulated control neutrophils. Neutrophils were activated with Mtb (MOI 10), PMA (25 nM) or left inactivated for 6 h before fixation. DNA was visualized with DAPI (blue), and Hsp72 was detected by antibody staining (red).

Fig. 7

Release of IL-6, TNF-α, IL-1β and IL-10 from macrophages stimulated with human recHsp72 (1 µg/ml). Neutrophils were activated with PMA (25 nM) for 3 h to form NETs, whereafter recHsp72 and macrophages were added to the wells, and cells were coincubated for 18 h. Mean values and error bars representing SEM from duplicates from 3 separate experiments are shown.* p < 0.05; ** p < 0.01.