Abstract
Exposure to widely used inert fibrous nanomaterials (for example, glass fibres or carbon nanotubes) may result in asbestos-like lung pathologies, becoming an important environmental and health concern. However, the origin of the pathogenesis of such fibres has not yet been clearly established. Here we report an electrochemical nanosensor that is used to monitor and quantitatively characterize the flux and dynamics of reactive species release during the frustrated phagocytosis of glass nanofibres by single macrophages. We show the existence of an intense prolonged release of reactive oxygen and nitrogen species by single macrophages near their phagocytic cups. This continued massive leakage of reactive oxygen and nitrogen species damages peripheral cells and eventually translates into chronic inflammation and lung injury, as seen during in vitro co-culture and in vivo experiments.
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Data availability
Source data are provided with this paper. All other data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.
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Acknowledgements
We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grants 22090050, 22090051, 21725504 and 21721005 to W.-H.H.; Grant 22122408 to Y.-L.L.; Grant 42221001 to P.F.) and the University of Xiamen and the State Key Laboratory of Physical Chemistry of Solid Surfaces (PCOSS, University of Xiamen, China) through an Outstanding Professor Chair awarded to C.A. In France, the work was supported in part by PSL University, École Normale Supérieure, CNRS and Sorbonne University (UMR 8640). Both teams gratefully acknowledge the CNRS Sino-French support (IRP ‘Nano-BioCatEchem’).
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C.A., Y.-T.Q. and W.-H.H. conceptualized the study and designed the research. Y.-T.Q. performed major experiments with the help of F.-L.Z., S.-Y.T., H.-Q.W., Y.Z., X.-W.Z., Y.-L.L. and P.F. Analysis of the data was by Y.-T.Q., C.A. and W.-H.H. with the help of F.-L.Z. and X.-W.Z. The paper was written by Y.-T.Q., C.A., W.-H.H. and P.F. All authors discussed the results and commented on the manuscript.
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Extended data
Extended Data Fig. 1 Morphology characterization, elemental analysis and endotoxin contamination of glass nanofibers.
a, SEM images of a glass nanofibers bundle and an individual long and short fiber. Scale bars, 10 μm (left and middle), and 2 μm (right). b, c, Representative histograms of the distributions and mean values of length, diameter and aspect ratio of long (b) and short (c) glass nanofibers. The measurements of aspect ratios were performed individually for each measured fiber. After eliminating a negligible number deviation, the remaining data were subjected to statistical analysis, which led to slightly different statistical mean values than when evaluating this value using the ratio between the statistical mean diameter and length (viz., 17 vs 15.5, respectively). d, Enlarged SEM image and EDX elemental mapping (Si and O) images of glass nanofibers used in this work. Scale bar,1 μm. e, Weight percentage of regular elements and soluble metals in glass fibers. f, Endotoxin content of 10 mg/mL glass fibers (n = 3 independent experiments; mean ± s.d.; one-way ANOVA); the glass fiber solution’s endotoxin content was significantly lower than the U.S. Pharmacopeia’s allowable limit of 0.25 EU/ml for endotoxin in water for injection.
Extended Data Fig. 2 The behavior of frustrated phagocytosis of glass fibers into macrophages.
a, Bright field and fluorescence microscopy images of RAW 264.7 cells stained with calcein-AM (green) and PI (red) before and after undergoing frustrated phagocytosis of glass nanofibers for 24 h. Scale bar, 50 μm. b, Cytotoxicity of macrophages as measured by cytosolic lactate dehydrogenase (LDH) released from fiber-damaged cells relative to M0 cell control (n = 3 biologically independent samples; mean ± s.d.; one-way ANOVA). c, d, Flow cytometer analysis on M1-like (CD86 and iNOS) and M2-like (CD206 and Arginase1) RAW 264.7 macrophages undergoing phagocytosis of long (c) and short glass (d) nanofibers. e, Individual Z-images of the Z-stacked confocal picture displayed in Fig. 1c. RAW 264.7 cells stained with phalloidin (orange) and Hoechst (blue) after undergoing frustrated phagocytosis of glass nanofibers (shown in green) during 12 h. Scale bars,10 μm. f, Morphology of macrophages stained with phalloidin (orange) and Hoechst (blue) after frustrated phagocytosis of a glass nanofiber (green) during 4, 12, 20, 24 h. Scale bar, 10 μm.
Extended Data Fig. 3 Principle of measurements of individual ROS/RNS fluxes.
a, Voltammetric oxidation of ONOO− (1 mM, pH=10.0), H2O2 (1 mM, pH=7.4), NO (1 mM, pH=7.4) and NO2− (1 mM, pH=7.4) in PBS solution as recorded with the present SiC@Pt NW sensor; currents were normalized to their maximum plateau value to help comparison. Vertical dashed lines indicate the optimal detection potentials for quantifying each ROS/RNS species (see Methods section). b, Staircase periodical potential sequence applied to the SiC@Pt NW sensor involving steps of 20 s duration at +150, +550, +650, and +800 mV vs. Ag/AgCl as indicated. c, Typical amperometric trace recorded upon applying the quadruple potential step chronoamperometric sequence described in Fig. 2a when a SiC@Pt NW sensor tip is moved from a position in solution (t < 160 s) to a position above a cell undergoing a frustrated phagocytosis (t > 160 s). The colored dots at the end of each 20 s long correspond to the sampling points at the end of each potential step. d, Sketch of the network of rapid reactions pathways relating the two precursors O2•- and NO to their follow-up primary products (boxes on the right-hand-side). O2•- and NO, the precursors of all ROS/RNS, are generated by NOX and iNOS, respectively. O2•- disproportionates into H2O2 (stoichiometry: 2O2•- per H2O2) either spontaneously (or through catalysis by superoxide dismutase within cellular bodies); O2•- reacts faster with NO to form ONOO−(stoichiometry: 1O2•- and 1NO per ONOO−) which is a transient species rapidly evolving into its end-product NO2− (whose formations thus corresponds also to 1O2•- and 1NO per NO2−). Note that NO also reacts with O2 to generate NO2−, however this occurs over longer times than the diffusional ones considered in this work.
Extended Data Fig. 4 ROS/RNS production rates of M0 macrophages.
a, Pseudo-color SEM image of one M0 macrophage (red shades). Scale bars, 2 μm. b, Time variations of the chronoamperometric currents measured when the at the tip of the nanosensor was located at the top of macrophage at each potential-step value: 150 (blue), 550 (gray), 650 (yellow), and 800 mV (green) vs. Ag/AgCl; scale bar, 10 µm. c, Corresponding time variations of ONOO− (blue), H2O2 (orange), NO (green), and NO2− (purple) production rates deduced from the currents in panel b. d, Time variations of the production rates of the two precursors O2•- (red) and NO (dark blue) as deduced from the data in panel c according to the stoichiometries reported in Extended Data Fig. 3d. caption. e, Statistical analyses (n = 6 macrophages analyzed over 40 min each) of the mean production rates of four primary ROS/RNS and their precursors O2•- and NO averaged over 40 min time windows (mean ± s.e.m.; one-way ANOVA). The vertical arrow in panels b-c corresponds to the moment when the SiC@Pt NW sensor was positioned at the top of the cell as shown in the inset in panel b.
Extended Data Fig. 5 Expression of iNOS during frustrated phagocytosis and current changes as nanosensor scanning the cell surface along the fiber axis after 12 h of frustrated phagocytosis.
a, Fluorescence images of macrophages stained with iNOS stained using its fluorescent-labelled monoclonal antibody (green), phalloidin (orange) and Hoechst (blue) after undergoing frustrated phagocytosis of glass nanofibers during 0, 4, 12, 20, 24 h. b, Statistical analysis (n = 90 cells from 3 independent samples) of the fluorescent intensity of stained iNOS (mean ± s.e.m.; one-way ANOVA). Scale bar, 100 μm. c, Sequence of bright-field microphotographs of the SiC@Pt NW sensor tip poised at +800 mV vs. Ag/AgCl during a scan along the cell surface following the glass nanofiber axis after 12 h of frustrated phagocytosis. Scale bar (see microphotograph 1): 20 μm. d, Currents intensities recorded at each position as indicated on panel a. Positions 4 and 16 marked by red circles are the locations of the two phagocytic cups.
Extended Data Fig. 6 Time variations of current and ROS/RNS generation rate of frustrated phagocytosis after 20 h.
a, Sequence of bright-field microphotographs of the SiC@Pt NW sensor with its tip poised at +800 mV vs Ag/AgCl during a scan along the cell surface following the glass nanofiber axis after 20 h of frustrated phagocytosis. Scale bar (see microphotograph 1): 20 μm. b, Currents intensities recorded at each position indicated on panel a. Position 4 marked by red circle is the location of the unsealed phagocytic cup; the absence of a significant current at position 30 indicates that the uropod extremity has been sealed. c, f, Time variations of the chronoamperometric currents measured at the phagocytic cup (c) and on the cell top (f) of a macrophage having undergone a frustrated phagocytosis of a glass nanofibers during 20 h when the nanosensor tip was poised at different potential values: 150 (blue), 550 (gray), 650 (yellow), and 800 mV (green) vs. Ag/AgCl. d, g, Corresponding time variations of ONOO− (blue), H2O2 (orange), NO (green), and NO2− (purple) production rates at the phagocytic cup (d) and at the cell top (g) as deduced from the currents in panel c and f, respectively. e, h, Time variations of the production rates of the two precursors O2•- (red) and NO (dark blue) at the phagocytic cup (e) and at the cell top (h) as deduced from the data in panel d and g, respectively, according to the stoichiometries reported in Extended Data Fig. 3d caption. The vertical arrow in each panel indicates the moment when the SiC@Pt NW tip was placed as indicated in the insets in panel c and f (scale bars, 20 μm).
Extended Data Fig. 7 Time variations of current and ROS/RNS generation rate of frustrated phagocytosis after 24 h.
a, Bright-field images sequence of the SiC@Pt NW sensor scanning along the cell surface following the glass nanofiber axis after 24 h of frustrated phagocytosis. Scale bar (see microphotograph 1), 20 μm. b, Variations of the electrochemical current with the location site of its tip polarized at +800 mV vs. Ag/AgCl. c, f, Time variations of the chronoamperometric currents measured at the cell extremity (c) and the cell top (f) after a macrophage underwent frustrated phagocytosis of a glass nanofiber during 24 h when the nanosensor tip was poised at 150 (blue), 550 (gray), 650 (yellow), and 800 mV (green) vs. Ag/AgCl. d, g, Corresponding time variations of ONOO− (blue), H2O2 (orange), NO (green), and NO2− (purple) production rates at the cell extremity (d) or on the cell top (g) as deduced from the currents in panels c and f, respectively. e, h, Time variations of the production rates of the two precursors O2•- (red) and NO (dark blue) at the cell extremity (e) or on the cell top (h) as deduced from the data in panel d and g, respectively, according to the stoichiometries reported in the caption of Extended Data Fig. 3d. Arrows indicate the time when the SiC@Pt NW sensor tip was positioned as indicated in the insets of panels c and f (scale bars, 20 μm).
Extended Data Fig. 8 ROS/RNS production rates of macrophages phagocytosis of short fibers.
a, Pseudo-color SEM images of macrophage (red) phagocytosis of a short glass nanofiber (blue) after 4 h; scale bars: 5 μm (main images), 1 μm (enlarged image), b-d, Time variations of the chronoamperometric currents (b), corresponding time variations of production rates of four primary ROS/RNS (c) and two precursors (O2•- and NO) (d) measured at the cell top of a macrophage after 4 h of attempted phagocytosis of short fibers. e, Statistical analyses (n = 6 macrophages analyzed over 1.5 h each) of the production rates of four primary ROS/RNS and of their precursors O2•- and NO after 4 h of attempted phagocytosis (mean ± s.e.m.; one-way ANOVA). f, Pseudo-color SEM images of macrophage (red) phagocytosis of a short glass nanofiber (blue) after 12 h; scale bars: 5 μm (main images), 500 nm (enlarged image). g, Z stacked confocal image of macrophages 12 h after beginning of phagocytosis of short glass nanofibers and its individual stacked Z-images. Scale bar, 10 μm. h-j, Time variations of the chronoamperometric currents (h), corresponding time variations of production rates of four primary ROS/RNS (i) and two precursors (j) measured at the cell top of a macrophage after 12 h of attempted phagocytosis of short fibers. k, Statistical analyses (n = 6 macrophages analyzed over 1.5 h each) of the production rates of four primary ROS/RNS and of their precursors after 12 h of attempted phagocytosis (mean ± s.e.m.; one-way ANOVA). l, Time variation of the production rates of the four primary ROS/RNS and of their two precursors released during phagocytosis of a short glass nanofiber. m, Same data as in panel l presented in terms of relative time-dependent proportions for the four primary ROS/RNS and their two precursors.
Extended Data Fig. 9 ROS/RNS production rates of LPS-stimulated macrophages and inflammatory response triggered by glass fibers.
a, Time variations of the chronoamperometric currents measured when the at the tip of the nanosensor was located at the pseudopodium of an LPS-stimulated macrophage at each potential-step value. b, Corresponding time variations of ONOO− (blue), H2O2 (orange), NO (green), and NO2− (purple) production rates of LPS-stimulated macrophages as deduced from the currents in panel a. c, Time variations of the production rates of the two precursors O2•- (red) and NO (dark blue) of LPS-stimulated macrophage as deduced from the data in panel b according to the stoichiometries reported in Extended Data Fig. 3d caption. d, Statistical analyses (n = 6 macrophages analyzed over 1.5 h each) of the mean production rates of four primary ROS/RNS and their precursors O2•- and NO of LPS-stimulated macrophages. (mean ± s.e.m.; one-way ANOVA). The vertical arrow corresponds to the moment when the SiC@Pt NW sensor was positioned at the pseudopod of the cell as shown in the inset in panel a. e, f, Time variation in the ratio of the production rates at the cup/extremity to those at the top of four primary ROS/RNS (e) and of their two precursors O2•- and NO (f) during frustrated phagocytosis of a long glass nanofiber. g, Heatmap of the cytokine array of macrophages with or without phagocytosis of long and short fibers after 24 h (n = 3 biologically independent samples; comparison of the significant differences between long and short fibers. mean ± s.d.; one-way ANOVA). The three columns shown for each category (viz., M0, short or long fibers) display the color outcomes of three different parallel experiments.
Extended Data Fig. 10 Co-culture and in vivo experiments.
a, Left: Schematic diagram showing the co-culture assay without RAW 264.7 cells; right: calcein-AM and PI fluorescent staining of MLE-12 cells with (bottom) or without (top) glass nanofibers; scale bar, 100 μm. b, Death rate of MLE-12 cells with or without glass fiber and RAW 264.7 macrophages (n = 3 biologically independent samples; mean ± s.d.; one-way ANOVA). c, Fluorescent images of alveolar macrophages recovered from bronchoalveolar lavage fluid stained with Siglec F (green), Hoechst and phalloidin (orange). Scale bar, 50 μm. d, Flow cytometer analysis on CD80 and CD86 of macrophages in BALF with or withour intratracheal instillation of long nanofiber (0.5 mg/rat) for 1day. e, Time-chart of the in vivo experiments. f, Bright-field and fluorescence microphotographs of alveolar macrophages stained with DCFH-DA or DAF-FM DA and Hoechst, with magnified images of single macrophages undergoing phagocytosis (yellow arrowheads) or co-phagocytosis (red arrowheads) of glass nanofibers (2 mg/rat) shown on the side; scale bars, 50 μm and 10 μm (magnified views). g, Representative hematoxylin and eosin (H&E) staining of lung sections with and without intratracheal glass nanofibers instillation (2 mg/rat) for 14 days; the magnified image shows the accumulation of glass nanofibers in the lung (top), and an alveolar macrophage pointed by the red arrow (bottom); scale bars, 100 μm (left), 20 μm (right top) and 5 μm (right bottom). h, Masson’s trichrome staining of lung sections with (bottom) and without (top) intratracheal glass nanofibers instillation (2 mg/rat); scale bar, 200 μm.
Supplementary information
Supplementary Information
Supplementary Tables 1–3 and Fig. 1.
Supplementary Video 1
Bright-field video of M0 macrophage proliferation.
Supplementary Video 2
Bright-field video of macrophage phagocytosis of glass nanofibre.
Supplementary Video 3
Confocal video of M0 macrophage proliferation.
Supplementary Video 4
Confocal video of macrophage phagocytosis of glass nanofibre.
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Qi, YT., Zhang, FL., Tian, SY. et al. Nanosensor detection of reactive oxygen and nitrogen species leakage in frustrated phagocytosis of nanofibres. Nat. Nanotechnol. 19, 524–533 (2024). https://doi.org/10.1038/s41565-023-01575-0
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DOI: https://doi.org/10.1038/s41565-023-01575-0
