Oligonucleotide sequences for the synthesis of DNase sensors that reports DNase recruitment in the macrophage phagocytic cups.

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) 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 sec after the PC formation indicated by F-actin signal.

DNase activity is universally present in the 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) 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)

DNase in the PCs was identified as membrane-bound DNaseX.

(A) F-actin and SNS signals in the PCs of THP-1 macrophages treated with PI-PLC, which cleaves 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 of 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 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.

DNaseX is constitutively recruited to the 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.

Distribution of DNaseX in the PCs, on the plasma membrane and inside the cell.

(A-B) 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 EDTA (A) or trypsin (B) prior to cell plating. The cells were incubated on the SNS surfaces for 1 h. (C-D) DNase activities in the PCs indicated by SNS signals with 20 min, 60 min 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)

F-actin structure is correlated with DNase activity but not with DNaseX recruitment in the PCs.

(A) Images of F-actin, DNaseX and SNS signals in the PCs with the macrophages treated with 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).

Macrophages degrade 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. (F) Analysis of eDNA degradation by macrophages. The degradation process typically spans 20-30 minutes.