A novel image-based quantitative method for the characterization of NETosis

Abstract

NETosis is a newly recognized mechanism of programmed neutrophil death. It is characterized by a stepwise progression of chromatin decondensation, membrane rupture, and release of bactericidal DNA-based structures called neutrophil extracellular traps (NETs). Conventional 'suicidal' NETosis has been described in pathogenic models of systemic autoimmune disorders. Recent in vivo studies suggest that a process of 'vital' NETosis also exists, in which chromatin is condensed and membrane integrity is preserved. Techniques to assess 'suicidal' or 'vital' NET formation in a specific, quantitative, rapid and semiautomated way have been lacking, hindering the characterization of this process. Here we have developed a new method to simultaneously assess both 'suicidal' and 'vital' NETosis, using high-speed multi-spectral imaging coupled to morphometric image analysis, to quantify spontaneous NET formation observed ex-vivo or stimulus-induced NET formation triggered in vitro. The use of imaging flow cytometry allows automated, quantitative and rapid analysis of subcellular morphology and texture, and introduces the potential for further investigation using NETosis as a biomarker in pre-clinical and clinical studies.

Keywords: Cell death; Flow cytometry; Microscopy; Neutrophil extracellular traps.

Figure 1. Quantification of suicidal NETosis using flow-based imaging and image analysis

a) Quantitative analysis of nuclear decondensation. Left panels show representative images from untreated (Un) or PMA-treated (PMA) neutrophils. Eight individual cells are shown by multispectral imaging of Brightfield (BF), side-scatter (SSC), and DNA staining (Hchst). Representative nuclei (yellow boxes) are detailed in the right panels where the nuclear mask (green) used for calculation of Nuclear Area (upper image) and Bright Detail Intensity (BDI, lower image) are shown for Untreated and PMA-treated samples. Values for Nuclear Area and BDI for the individual represe ntative images are shown inset in yellow text. b) A region containing cells with high nuclear area and low BDI is shown in red (left dotplots) for Untreated (upper panels) or PMA-treated (lower panels) samples. Arrows indicate the data points of the single representative cells shown in a). Cells in the gated region (red dots) are shown backgated onto dotplots of SSC and Hoechst Intensity (right dotplots). Scale bars are 7μm²

Figure 2. Myeloperoxidase (MPO) colocalization with DNA in decondensed nuclei following LPS stimulation

a) Eight representative cells with normal (upper panels) or decondensed (lower panels) nuclei from untreated (Un) or LPS-treated (LPS) neutrophils are shown, with BF, MPO, Hoechst (Nuc), and an overlay of Hoechst and MPO (merge) images indicated. Scale bars are 7μm. b) Quantification of the Nuclear Area and BDI is shown in the dotplots for Untreated (top) and LPS-treated (bottom) samples. Nuclei with normal Nuclear Area and BDI are gated in blue, and those with high Nuclear Area and low BDI are gated in red. Right histogram overlay shows negative correlation of MPO and DNA localization for normal nuclei (blue trace) but positive correlation of MPO and DNA localization in decondensed nuclei (red trace) by log-transformed Pearson’s correlation coefficient (Similarity).

Figure 3. Observation and quantification of morphology change following LPS stimulation

a) Six representative BF images of Untreated and LPS-treated samples are shown. b) Hand-selected images of cells with normal, round appearance (magenta dots) and those of elongated cells with membrane protrusions (blue dots) are plotted using the image analysis features that best distinguish these morphologies, determined by Fisher’s Discriminate Ratio (RD); BF Aspect Ratio and Delta Centroid XY BF, BF Intensity. Images to the right of the dotplots illustrate how these measurements are made. c) Eight representative images of cells in BF, SSC, Hoechst (Nuc) and an overlay of those images (merge) are displayed for the normal, round morphology of Untreated cells and for the elongated morphology observed following LPS-treated cells. Feature values for Aspect Ratio and Delta Centroid XY are displayed in blue text on the BF and merge images, respectively. d) Dotplot showing the appearance of cells with low Aspect Ratio and high Delta Centroid XY (red dots) after LPS treatment. Cells with elongated morphology (region outlined in cyan) are shown backgated onto the dotplot of Nuclear Area versus Hoechst Bright Detail Intensity (cyan spots), and have those features in common with normal neutrophil nuclei (see Figure 1b).

Figure 4. Myeloperoxidase (MPO) does not colocalize with DNA in elongated cells after LPS treatment

Nine representative individual elongated cells are shown to demonstrate anti-correlation between DNA and MPO (Hchst/MPO) in this phenotype. Cell surface and internalized sialic acid and N-acetylglucosamine are labeled with Wheat Germ Agglutinin (WGA) in order to visualize the plasma membrane. Anti-correlation between DNA and MPO (Similarity MPO, Hoechst) in elongated cells (blue trace), is shown relative to the positive correlation between DNA and MPO among decondensed nuclei (red trace), and the anti-correlation between those in normal nuclei (green trace). b) Three individual cells are shown exhibiting only WGA staining and very little BF contrast, Hoechst, and MPO positivity. These cells also displayed very low or no SSC. Scale bars are 7μm.

Figure 5. Features of ‘vital” NETosis are not inhibited by pan-caspase inhibitors

Neutrophils were pre-treated with the pan-caspase inhibitor Z-VAD-FMK (20 mM) for 30 minutes, followed by stimulation with LPS for an additional hour. Results represent mean % of cells undergoing ‘vital’ or ‘suicidal’ NETosis in the presence of LPS+/− DMSO or vehicle.

Figure 6. Comparisons between induction of ‘vital’ versus ‘suicidal’ NETosis features in control and lupus neutrophil subsets

Untreated neutrophils from healthy control, normal and low density neutrophils from lupus patients (a,n=6), treated with PMA for 4 hours (b,n=3) or LPS for 1 hour (c,n=5) were stained with Hoechst 33342. Suicidal and Vital NETosis rate was quantified using the method described above.