Early recruitment of membrane-bound DNaseX to phagocytic cups in macrophages
- Hoxworth Center, College of Medicine, University of Cincinnati, United States
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati, United States
Figures
Figure 1 with 8 supplements
Early onset of DNase activity in the phagocytic cup (PC) prior to its closure.
(A) Surface-immobilized nuclease sensor (SNS) reports DNase activity in the PCs of macrophages. Microbeads are immobilized on a glass substrate to elicit phagocytosis. The entire surface, including the microbeads, is coated with SNS, a double-stranded DNA (dsDNA) labeled with a quencher-dye pair. SNS becomes fluorescent upon degradation by DNase. (B) Human THP-1 macrophages were plated on the DNase-reporting platform, where fluorescence signals appeared in ring patterns, co-localizing with F-actin surrounding the microbeads. (C) Comparison of SNS fluorescence intensities on microbeads underneath cell bodies versus those outside of cells. (****p<0.0001; Each data point represents one PC; n=3 experiments; error bars indicate SD.) (D) Confocal 3D scanning revealed that the SNS signal was localized on the surface of microbeads. (E, F) dsDNA-dye coated on microbeads exhibited a decrease in fluorescence intensities beneath macrophages. (G, H) Single-stranded DNA (ssDNA)-dye coated on microbeads exhibited a decrease in fluorescence intensities beneath macrophages. (I) Time-lapse co-imaging of F-actin and SNS signals in the PCs of live macrophages. (J) Signal intensity curves of F-actin and SNS on one microbead. The time gap Δt between the starting times of these two signals is defined as the emergence time of DNase activity in the PCs. (K) Emergence time of DNase activity in the PCs was statistically estimated to be 48±33 s after the PC formation indicated by F-actin signal.
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Figure 1—source data 1
Numeric data and statistical analysis for the plots in Figure 1 and associated figure supplements.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Surface-immobilized nuclease sensor (SNS) immobilized on glass surfaces responds to the treatment of soluble DNase I.
(A) 1 U/mL DNase I (Thermo Fisher Scientific, #89836) in Tris buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, pH 7.5) was loaded on an SNS-coated glass surface. Fluorescence images were acquired as a time series. (B) SNS response curves to the treatment of DNase I at concentrations of 0 (control), 1 U/mL, and 10 U/mL.
Figure 1—figure supplement 2
Surface-immobilized nuclease sensor (SNS) signals on microbeads specifically occurred underneath macrophage cell bodies.
The SNS sensor displays weak fluorescence despite the quencher in the sensor construct for the non-100% quenching efficiency. This low background fluorescence was leveraged to visualize all microbeads on the glass surface by digitally enhancing the image contrast. It was confirmed that the SNS signals, indicated by localized increases in fluorescence, appeared exclusively on the microbeads located beneath the cell bodies of THP-1 macrophages. This observation reinforces the notion that the DNase activity indicated by SNS signals is specific to the cell regions. The yellow contour represents the cell’s edge.
Figure 1—figure supplement 3
Large-area imaging of THP-1 macrophages on a surface-immobilized nuclease sensor (SNS) surface.
Fluorescent beads with an excitation peak at 405 nm were used in SNS experiments to facilitate their localization during fluorescence imaging. Large-area imaging was performed over human THP-1 macrophages plated on an SNS-coated surface, showing microbeads (λ=405 nm, blue), F-actin (λ=488 nm, green), and SNS-reported DNase activity (λ=647 nm, magenta). The macrophages were incubated on the surface for 1 hr prior to cell fixation and staining. These images demonstrate that THP-1 macrophages specifically and consistently produce DNase activity in phagocytic cups.
Figure 1—figure supplement 4
Large-area imaging of RAW macrophages on a surface-immobilized nuclease sensor (SNS) surface.
Large-area imaging was performed over mouse RAW 264.7 macrophages plated on an SNS-coated surface, showing microbeads (λ=405 nm, blue), F-actin (λ=488 nm, green), and SNS-reported DNase activity (λ=647 nm, magenta). The macrophages were incubated on the surface for 1 hr prior to cell fixation and staining. These images demonstrate that RAW macrophages specifically and consistently produce DNase activity in phagocytic cups.
Figure 1—figure supplement 5
The DNase in phagocytic cups (PCs) degrades plasmid DNA.
(A) 50 µg/mL plasmid DNA was immobilized on a microbead-decorated and poly-L-lysine (PLL)-coated glass surface. The immobilization of plasmid DNA was enabled by the electrostatic force as the PLL coating is positively charged and the DNA backbone is negatively charged. The DNA was stained with SYTOX Green. RAW macrophages were incubated on the surface for 1 hr. The sample was directly imaged without cell fixation or staining. (B) Fluorescence intensities of SYTOX Green on microbeads. The microbeads underneath cells had lower fluorescence intensities than those out of cells, suggesting that PCs degrade plasmid DNA as well.
Figure 1—figure supplement 6
F-actin and surface-immobilized nuclease sensor (SNS) signal intensities in individual phagocytic cups (PCs).
F-actin and SNS signal intensity curves from six additional PCs. Imaging was performed in live macrophages transfected with LifeAct-GFP and plated on SNS-coated surfaces. Related to Video 1.
Figure 1—figure supplement 7
Surface-immobilized nuclease sensor (SNS) signal intensities in the phagocytic cups (PCs) after cell plating for 5, 10, and 30 min, respectively.
(A) Co-imaging of F-actin and SNS in the PCs of THP-1 macrophages with 5, 10, and 30 min cell incubation times. (B) Quantification of the SNS signal intensities. Each data point represents the total fluorescent intensity of the SNS signal on a microbead. ****p<0.0001. Each data point represents the SNS signal in one PC.
Figure 1—figure supplement 8
The phagocytic cup (PC) exhibits DNase activity over free microbeads (not surface-immobilized).
Streptavidin-functionalized microbeads (3 µm) were coated with surface-immobilized nuclease sensor (SNS) via the avidin-biotin interaction and then added to surface-adherent RAW macrophages. After 30 min of incubation, many microbeads (red arrows) had been internalized by the macrophages and exhibited strong fluorescence, indicating DNase activity within the phagosomes. Several microbeads appeared to be undergoing phagocytosis, which is not yet completed and were co-localized with PCs, as indicated by F-actin-rich cup structures. Notably, these microbeads (yellow arrows) displayed non-uniform fluorescence across their surfaces, suggesting that PCs already have DNase activity in response to free microparticles before PC closure.
Figure 2 with 1 supplement
DNase activity is universally present in the phagocytic cups (PCs) of various macrophage types.
(A–C) DNase activities were consistently observed in the PCs of mouse RAW macrophages, human THP-1 macrophages, and human monocyte-derived macrophages. The PCs, marked by F-actin structures, were formed on microbeads of various sizes (0.3, 1.1, and 3.0 µm). (D) DNase activity was consistently observed in the PCs of M0, M1, and M2 THP-1 macrophage subtypes. (E–G) Surface-immobilized nuclease sensor (SNS) signal intensities in the PCs of the three types of macrophages. (Each data point represents one PC; n=3 experiments; error bars indicate SD.) (H) SNS signal intensities in the PCs of the three subtypes of THP-1 macrophages. (Each data point represents one PC; n=3 experiments; error bars indicate SD.)
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Figure 2—source data 1
Numeric data and statistical analysis for the plots in Figure 2.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Human monocyte extraction from white blood cell (WBC)-enriched blood sample.
(A) A leukocyte reduction system (LRS) chamber used during blood donation was collected. The chamber contains WBC-enriched blood sample, providing a source for extracting human monocytes. The blood sample was drained into a 50 mL tube. Phosphate-buffered saline (PBS) supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA) and 2% fetal bovine serum (FBS) was added to the blood sample at a volumetric ratio of 3:1 (PBS:blood). (B) 6 mL diluted blood sample was loaded on top of 3 mL gradient medium (the colorless transparent liquid at the bottom of the tube) carefully, forming a clear line between two liquids. The sample was centrifuged at 400×g for 15 min. Consequently, red blood cells were spun to the bottom, and peripheral blood mononuclear cells (PBMCs) were concentrated between the plasma (the pinkish top layer of liquids) and the gradient medium. The PBMCs were collected by pipetting and suspended in 2 mL PBS supplemented with 2 mM EDTA and 2% FBS. (C) Monocytes in the PBMC solution were isolated using a negative selection kit. This kit employs antibody-coated paramagnetic beads to capture all non-monocyte cells, leaving monocytes in the solution. The purified monocytes were subsequently centrifuged and suspended in culture medium for cell culture and experimentation, or, alternatively, suspended in a solution of 20% dimethyl sulfoxide (DMSO) and 80% FBS for cryogenic preservation.
Figure 3 with 2 supplements
DNase in the phagocytic cups (PCs) was identified as membrane-bound DNaseX.
(A) F-actin and surface-immobilized nuclease sensor (SNS) signals in the PCs of THP-1 macrophages treated with phosphoinositide phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol (GPI) linkers of the putative membrane-bound DNase in the PCs. (B) PI-PLC treatment significantly reduced SNS signals in the PCs, indicating marked decreases in DNase activities. (****p<0.0001; Each data point represents one PC; n=3 experiments; error bars indicate SD.) (C) F-actin and SNS signals in the PCs of THP-1 macrophages with DNaseX knocked down by small interfering RNA (siRNA) interference. (D) DNaseX knockdown significantly reduced SNS signals in the PCs. (****p<0.0001; Each data point represents one PC; n=3 experiments; error bars indicate SD.) (E) Co-imaging of immunostained DNaseX, F-actin, and SNS signals in the PCs of THP-1 macrophages.
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Figure 3—source data 1
Numeric data and statistical analysis for the plots in Figure 3.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Gene expression of DNaseX (a.k.a. DNase I-like 1) in human hematological cells.
The gene expression levels of DNaseX in human hematological cells are available in the Expression Atlas, a comprehensive database maintained by the European Bioinformatics Institute (EBI) that catalogs gene expression across various biological conditions and tissues. According to the data, mRNA of DNaseX is expressed at higher levels in macrophages and monocytes compared to other hematological cells, whereas mRNA of DNase I shows lower expression levels in macrophages relative to other hematological cell types.
Figure 3—figure supplement 2
DNaseX was recruited onto microbeads beneath the domes of phagocytic cups (PCs).
(A) Co-imaging of DNaseX (immunostained), F-actin, and surface-immobilized nuclease sensor (SNS) in a THP-1 macrophage. (B) 3D scan of DNaseX, F-actin, and SNS using confocal microscopy. All three signals are shown to be on the microbeads beneath the domes of the PCs, not on the substrate regions around microbeads.
Figure 4 with 3 supplements
DNaseX is constitutively recruited to the phagocytic cups (PCs) without requiring the presence of DNA materials.
(A) DNaseX in the PCs in response to surface-immobilized E. coli, or microbeads coated with various biomaterials, including lipopolysaccharide (LPS), immunoglobulin G (IgG), fibronectin (FN), and poly-L-lysine (PLL). DNaseX was immunostained with antibodies for imaging. (B) Line profiles show the co-localization of DNaseX and F-actin in the PCs on these five surfaces. Yellow lines in (A) mark the locations for the line profile analysis.
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Figure 4—source data 1
Numeric data for the plots in Figure 4.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
DNaseX was recruited to the phagocytic cups (PCs) in response to surface-immobilized E. coli.
(A) Co-imaging of E. coli, F-actin, and immunostained DNaseX underneath a THP-1 macrophage. (B) Line profiles of E. coli, F-actin, and immunostained DNaseX. The region of analysis is marked with a red line in (A).
Figure 4—figure supplement 2
DNaseX is found in both plasma membrane and inside cells.
(A) Confocal scan of DNaseX on the plasma membrane of macrophages, which were detached by ethylenediaminetetraacetic acid (EDTA) and not permeabilized during cell fixation. The red dashed line indicates the section of 3D imaging displayed in the top view. (B) Confocal scan of DNaseX on the plasma membrane of macrophages, which were detached by trypsin and not permeabilized during cell fixation. (C) Confocal scan of DNaseX on the plasma membrane and in the cytoplasm of macrophages, which were detached by EDTA and permeabilized during cell fixation. (D) Confocal scan of DNaseX on the plasma membrane and in the cytoplasm of macrophages, which were detached by trypsin and permeabilized during cell fixation.
Figure 4—figure supplement 3
No co-localization between DNaseX and lipid rafts on the cell membrane of a macrophage.
Confocal scanning reconstructed the three-dimensional distribution of lipid rafts and DNaseX clusters on the plasma membrane of a macrophage. No apparent co-localization was observed between these two signals. The lipid rafts were stained with a lipid raft labeling kit (V-34405, Molecular Probes). Related to Video 2.
Figure 5
Distribution of DNaseX in the phagocytic cups (PCs), on the plasma membrane and inside the cell.
(A, B) Surface-immobilized nuclease sensor (SNS) signal, F-actin, and immunostained DNaseX on the ventral surfaces of adherent THP-1 macrophages, which were detached from a culture flask by either ethylenediaminetetraacetic acid (EDTA) (A) or trypsin (B) prior to cell plating. The cells were incubated on the SNS surfaces for 1 hr. (C, D) DNase activities in the PCs indicated by SNS signals with 20, 60, and 90 min incubation times, respectively. The macrophages were detached by either EDTA (C) or trypsin (D). (E) SNS signal intensities in PCs. (*p<0.05; ns p>0.05; Each data point represents one PC; n=3 experiments; error bars indicate SD.)
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Figure 5—source data 1
Numeric data for the plots in Figure 5.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig5-data1-v1.xlsx
Figure 6
F-actin structure is correlated with DNase activity but not with DNaseX recruitment in the phagocytic cups (PCs).
(A) Images of F-actin, DNaseX, and surface-immobilized nuclease sensor (SNS) signals in the PCs with the macrophages treated with dimethyl sulfoxide (DMSO) (control), 100 µM CK666, or 1 µM cytochalasin D, respectively. (B–D) Signal intensities of F-actin, SNS, and DNaseX signals in the PCs with the above treatments. (****p<0.0001; **p<0.01; Each data point represents one PC; n=3 experiments; error bars indicate SD).
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Figure 6—source data 1
Numeric data and statistical analysis for the plots in Figure 6.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig6-data1-v1.xlsx
Figure 7
Macrophages degrade extracellular DNA (eDNA) in biofilms by physical contact.
(A) eDNA structures in S. aureus biofilms. The eDNA was stained with DITO-1. (B) RAW macrophages incubated on S. aureus biofilms for 30 min. The filamentous eDNA structures were degraded under cell bodies. (C) Line profile analysis of eDNA degradation regions indicated by blue lines in (B), which are shown to have sharp boundaries with ~1 µm transition from undegraded region to degraded region, suggesting that the eDNA degradation was mediated by non-diffusive DNase. (D) Time-series images of eDNA degradation by a RAW macrophage. Related to Video 3. The eDNA filament (indicated by a blue arrow) became gradually shortened during the degradation. (E) Analysis of eDNA degradation by macrophages. The degradation process typically spans 20–30 min.
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Figure 7—source data 1
Numeric data for the plots in Figure 7.
- https://cdn.elifesciences.org/articles/110907/elife-110907-fig7-data1-v1.xlsx
Videos
Video 1
Co-imaging of surface-immobilized nuclease sensor (SNS) signals and F-actin in phagocytic cups.
The cells are RAW macrophages transfected with LifeAct-GFP. The actual duration of cell activity in the video is 30 min.
Video 2
3D reconstruction of DNaseX and lipid raft imaged in a THP macrophage.
Video 3
Macrophages degrading extracellular DNA (eDNA) structure in bacterial biofilm.
The biofilm was formed by S. aureus. Cells are RAW macrophages. The actual duration of cell activities in the video is 1 hr.
Tables
Table 1
Oligonucleotide sequences for the synthesis of DNase sensors that report DNase recruitment in the macrophage phagocytic cups.
| DNA strands for DNase sensor | Oligonucleotide sequences |
|---|---|
| Upper DNA strand for SNS | 5′-GGGCGGCGACCTCAGCAT/3BHQ_2/3′ |
| Lower DNA strand for SNS | 5′-/5BiosG/T/iAtto647N/ATGCTGAGGTCGCCGCCC-3′/ |
| Upper strand for dye-labeled dsDNA | 5′-GGGCGGCGACCTCAGCAT-3′ |
| Lower strand for dye-labeled dsDNA | 5′-/5BiosG/ATGCTGAGGTCGCCGCCC/Atto647N/-3′ |
| Dye-labeled ssDNA | 5′-/5BiosG/ATGCTGAGGTCGCCGCCC/Atto647N/-3′ |
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Early recruitment of membrane-bound DNaseX to phagocytic cups in macrophages
eLife 15:RP110907.
https://doi.org/10.7554/eLife.110907.3
