Detection of neutrophil extracellular traps in patient plasma: method development and validation in systemic lupus erythematosus and healthy donors that carry IRF5 genetic risk

Abstract

Neutrophil extracellular traps (NETs) are web-like structures extruded by neutrophils after activation or in response to microorganisms. These extracellular structures are decondensed chromatin fibers loaded with antimicrobial granular proteins, peptides, and enzymes. NETs clear microorganisms, thus keeping a check on infections at an early stage, but if dysregulated, may be self-destructive to the body. Indeed, NETs have been associated with autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), antiphospholipid syndrome (APS), psoriasis, and gout. More recently, increased NETs associate with COVID-19 disease severity. While there are rigorous and reliable methods to quantify NETs from neutrophils via flow cytometry and immunofluorescence, the accurate quantification of NETs in patient plasma or serum remains a challenge. Here, we developed new methodologies for the quantification of NETs in patient plasma using multiplex ELISA and immunofluorescence methodology. Plasma from patients with SLE, non-genotyped healthy controls, and genotyped healthy controls that carry either the homozygous risk or non-risk IRF5-SLE haplotype were used in this study. The multiplex ELISA using antibodies detecting myeloperoxidase (MPO), citrullinated histone H3 (CitH3) and DNA provided reliable detection of NETs in plasma samples from SLE patients and healthy donors that carry IRF5 genetic risk. An immunofluorescence smear assay that utilizes only 1 µl of patient plasma provided similar results and data correlate to multiplex ELISA findings. The immunofluorescence smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes of patient plasma.

Keywords: ELISA; NETosis; immunofluorescence; quantification; smear assay.

Figure 1

Higher NETs are detected in plasma from PMA treated samples and SLE samples using Sytox Green assay. Plasma samples were incubated with Sytox Green for 5 minutes and fluorescence was measured using microplate reader. (A) Plasma samples from untreated healthy controls (HC) and PMA treated healthy donors (PMA, n=7), (B) SLE patients (n=40) and healthy controls (n=20), and (C) non-risk (NR, n=12) and risk (R, n=10) donors from the Genotype and Phenotype Registry. Single data points represent individual donors. Plotted data are after background subtraction. Data are presented as mean ± SD. P values are reported after unpaired parametric T test was performed *<0.05; **<0.01.

Figure 2

Detection of NETs using different ELISA methodologies with plasma samples from Healthy donors, PMA stimulated donors, SLE patient, and non-risk and risk GaP patient samples. (A) ELISA plate coated with 5µg/ml NE (SantaCruz) antibody using Healthy (HC) and PMA stimulated plasma samples (n=7). (B) ELISA plates were coated with CitH3 antibody (Abcam) or combination of MPO (Abcam) and CitH3 (Abcam) antibodies at a concentration of 5µg/ml to test healthy donors (n=20) against SLE patient donors (n=40), (C) non-risk (n=12) and risk (n=10) associated samples from the Genotype and Phenotype registry, and (D) healthy donor against PMA stimulated plasma samples (n=7). ELISA was blocked using additional 5%NRS in the buffer and developed using TMB substrate and results demonstrate differences in plate absorbance values in the presence and absence of MPO (Abcam) in the initial antibody coating (B-D). (E) Extrapolated data from standard curve to represent DNA-MPO-CitH3 NET content in healthy controls (n=15) and SLE (n=37) and (F) GaP non-risk (n=10) and risk (n=7) samples using data from MPO+CitH3+DNA ELISA. Single data points represent individual donors. Plotted data are after background subtraction. Data are presented as mean ± SD. P values are reported after unpaired parametric T test was performed. **<0.01; ***<0.001; ****<0.0001.

Figure 3

Sytox Green and DAPI staining of PMA stimulated and unstimulated samples to confirm presence of circulating NETs. Plasma smeared poly-l-lysine slides were prepared and stained with Sytox Green and DAPI for (A) unstimulated healthy controls and (B) PMA stimulated controls. Overlap of DAPI and Sytox Green channels confirm presence of circulating NETs in PMA stimulated donors. Individual merged images represent separate donors (n=4). All images were taken on ZEISS Confocal M880 at 20x objective. Set scale 200µm.

Figure 4

Smear assay showing higher NETs in plasma from PMA stimulated samples, SLE samples, and risk GaP patient samples when compared to healthy donors. Representative images of plasma smeared poly-l-lysine slides stained with MPO (green, AF488), CitH3 (red, AF594) and DAPI (Blue). (A) Untreated healthy plasma smears display lower concentration of circulating NETs than (B) plasma of PMA stimulated samples. (C) Last image of PMA stimulated sample zoomed in 5x to show differential staining of MPO (green) and CitH3 (red) antibodies. (D) Healthy donor and (E) non-risk donor samples also show lower concentration of circulating NETs than (F) plasma of SLE and (G) IRF5 homozygous risk donors. Individual merged images represent separate donors (n=18). All images were taken on ZEISS Confocal M880 at 20x objective. Set scale 100µm.

Figure 5

Plasma smear circulating NETs can be detected through brightfield microscopy. Plasma smeared poly-l-lysine slides were prepared and stained with MPO antibody (green, AF488), CitH3 (red, AF594) and DAPI (blue) and single channel images were obtained for (A) unstimulated healthy control and (B) PMA stimulated samples. NETs were identified and brightfield images overlapped with specific antibody markers in (B) PMA stimulated sample, therefore confirming the presence of circulating NETs. Individual merged images represent separate donors (n=2). All images were taken on ZEISS Confocal M880 at 20x objective. Set scale 100µm.

Figure 6

Plasma smear quantification using 20x objective and average threshold-area pixel intensity. Using ImageJ, representative plasma smear images were converted to 8-bit grayscale images and pixel intensity threshold range was set from 40-255. Images were measured and averages of threshold-area pixel intensity were computed for (A) healthy control and PMA stimulated (n=5), (B) healthy control and SLE (n=5), and (C) GaP risk and non-risk (n=5). Observed differences between sample groups correlate to previously calculated NET quantity in ELISA. Data are presented as mean ± SD. P values are reported after unpaired parametric T test was performed. **<0.01.

Figure 7

Correlation analysis between MPO + CitH3 + DNA ELISA and plasma smear assay across patient samples. Quantity of circulating NETs in healthy control (n = 9) and SLE (n=5) patient samples were measured using the two different assays, and the correlation between assay results was determined. Single data points represent individual donors. Two-tailed correlation analysis between data sets was calculated using Pearson correlation coefficients assuming Gaussian distribution in GraphPad Prism 8. R² and p-value of linear regression are reported above.