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Vidhya Rao, Christine Mun, Sandeep Jain, Simon Kaja; Pooled human immune globulins protect against neutrophil activation. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1980 – A0310.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies have demonstrated the presence of anti-citrullinated protein autoantibodies (ACPA) in ocular surface washes from patients with autoimmune dry eye disease (DED). Moreover, autoimmune DED was responsive to treatment with pooled human immune globulin (pooled IgG) eye drops. The purpose of this study was to develop a plate reader-based in vitro assay to evaluate therapeutics targeting neutrophil activation and neutrophil extracellular traps formation (NETosis). Specifically, the efficacy of pooled IgG in preventing neutrophil activation and NETosis were assessed.
Primary human neutrophils (HemaCare) were maintained in phenol red-free RPMI1640 media supplemented with 10% fetal bovine serum. 1 × 105 neutrophils were seeded into black/ clear bottom 96-well plates (FIAv plates, Greiner Bio) and stained with 1 µM SYTOX green (S7020, Thermo Fisher Scientific), a fluorescent dye that is impermeant to live cells and has a high affinity to nucleic acid. Subsequently, cells were allowed to settle for 20 min and subsequently stimulated with either phorbol 12-myristate 13-acetate (PMA; 100 nM; 79346, Sigma-Aldrich), or anti-histone 4 R3 ACPA (H4R3 ACPA; 100 ng/mL; Abcam, #ab81797). Fluorescence of extracellular NET-bound SYTOX Green (excitation λ: 488 nm, emission λ: 510 nm) was measured every 30 min for a period of 72 h using a microplate reader (Cytation 5, Biotek Instruments) at 37°C. Dose-responses (0.001 – 4%) of pooled human IgG diluted in saline (Flebogamma) were tested for the ability to inhibit NETosis.
H4R3 ACPA resulted in ~2-fold increased SYTOX Green fluorescence over a 72 h period indicating neutrophil activation and NETosis. The level of NETosis elicited by H4R3 ACPA was similar to activation by PMA. Pooled IgG resulted in a dose-dependent decrease in SYTOX Green fluorescence that could be fitted with a fourth parameter non-linear regression (R2 = 0.95). 1% pooled IgG reduced SYTOX Green fluorescence to 115.4 ± 10.9% (mean ± S.D.) of control for H4R3 ACPA-induced NETosis and 99.6 ± 9.1 % (mean ± S.D.) for PMA-induced NETosis.
The plate reader-based SYTOX Green assay provides a standardized platform for testing NETosis-targeted therapeutics. Pooled IgG exert potent protection against ACPA-induced NETosis in vitro. Our findings confirm observations from our previous studies that have shown improvement of ocular surface pathology following Pooled IgG administration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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