Fig 1.
Microscopic examination of macrophage extracellular trap (MET) formation induced by M. mass infection.
Differentiated THP-1 macrophages were infected with M. mass R or CIP at a MOI of 5 for 24 hr, and stained by DNA-binding dye, TO-PRO-3 (Red) to visualize extracellular trap-like structure. (A) Microscopic examination of extracellular trap-like structures in M. mass R-infected THP-1 macrophages. Bar, 200μm. (B, C) Microscopic examination (B) and TO-PRO staining (C) of extracellular trap-like structures induced by M. mass R infection. Bar, 50μm. (D, E) Fluorescence micrographs of CFSE-stained M. mass R (Green) entrapped by METs. Bar, 20μm. (F) Scanning electron microscopy (SEM) images of METs induced by M. mass CIP. Bar, 1μm. Arrow: Mycobacteria. Arrow heads: Extracellular trap-like structures.
Fig 2.
Rough strain of M. mass strongly induces MET formation in differentiated THP-1 macrophages.
(A) Differentiated THP-1 macrophages were stimulated with M. mass R (MOI 20) with or without DNase I (50 units/ml), M. mass CIP (MOI 20), LPS (10 μg/ml) or hydrogen peroxide (1 mM) for 24 hr, and then stained by TO-PRO-3 to examine METs. Bar, 50μm. (B) MET formation (%) of each sample was quantified by calculating percentages of MET-positive cells to total cells count. MR: M. mass R. Data are representative of three independent experiments with similar results. *, p<0.05; **, p<0.01 compared to M. mass R-infected group by one-way ANOVA with Bonferroni’s post-test.
Fig 3.
M. mass R-induced METs induce release of mitochondrial as well as nuclear DNA, and contain Histone, MPO and Elastase.
(A) PCR analysis was performed for nuclear (β-actin, Gapdh) and mitochondrial genes (Atp6, Nds1) using DNA isolated from the supernatants of M. mass R-infected THP-1 macrophages and uninfected control samples. Both nuclear and mitochondrial genes were detected in the infected samples. Data are representative of three independent experiments. (B) M. mass R-infected THP-1 macrophages were fixed and processed for histone 4, elastase and MPO staining. METs were stained by TO-PRO-3. Circles indicate co-localization of METs with each component. Data are representative of three independent experiments. H4: histone 4. Bar, 20μm.
Fig 4.
M. mass R-induced MET formation is not dependent on NADPH oxidase activity, but does depend on calcium influx.
After staining THP-1 macrophages with TO-PRO-3, the MET formation (%) in each sample was determined by quantification of MET-positive cells to total cell count. (A) Differentiated THP-1 macrophages were stimulated with hydrogen peroxide (1 mM), M. mass R (MOI 5) with or without DPI (20 μM) for 24 hr. (B) Fluo-4-labeled THP-1 macrophages were untreated or pretreated with BAPTA (20 μM) or EGTA (1 mM) for 30 min and then infected with M. mass R (MOI 5). Ionomycin (1 μM) was used as positive control. Intracellular calcium transients of labeled cells were recorded in a microplate reader at 6 hr after infection. (C) Determination of MET formation (%] in each sample. MR: M. mass R. Data are representative of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001 compared to M. mass R-infected group by one-way ANOVA with Bonferroni’s post-test.
Fig 5.
MET formation is dependent on phagocytosis and cell lysis.
(A) CFUs of M. mass R recovered from infected macrophages with or without cytochalasin D (5 μM) for 24 hr. (B) MET formation (%) in the macrophages infected with M. mass R (MOI 10) after pretreatment with or without cytochalasin D. (C) CFUs recovered from macrophages infected with M. mass R at various MOI at 4 hr post infection. (D) Determination of MET formation (%) in each sample at 24 hr post infection. (E) Supernatants were collected from macrophages infected with M. mass R at various MOIs at 24 hr post infection. The level of cell lysis of each group was determined by LDH release assay. MR: M. mass R. Data are representative of three independent experiments. ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001 by Student’s t-test (A) or one-way ANOVA with Bonferroni’s post-test (B-E).
Fig 6.
Released METs have no bactericidal effects on M. mass R.
To determine the bactericidal effect of METs on M. mass R, macrophages with METs-degrading DNase I 30 min before infection. (A) Cell-associated (intracellular or METs-bound), (B) Extracellular, (C) Total number of bacteria recovered from the samples infected by M. mass R (MOI 5) for 24 hr, with or without DNase (50 units/ml). (D) AFB staining of THP-1 macrophages infected by M. mass R with or without DNase for 24 hr. Bar, 20μm. Data are representative of three independent experiments with similar results. **, p<0.01 by Student’s t-test.
Fig 7.
METs formation mediated by entry of M. mass into macrophage.
(A) Phagocytosis of M. mass by macrophages induces an increase in the level of intracellular calcium. Then, accompanying necrosis, METs are released and prevent bacterial dissemination by entrapping M. mass. The METs comprise of histones and antimicrobial enzymes (elastase and MPO) but rather facilitate bacterial survival and growth without bactericidal effects. (B) Treatment with cytochalasin D (Cyt D) prevents phagocytosis of M. mass by macrophage, thereby resulting in decreased METs production.