Abstract
Purpose :
Differentiated HL-60 (dHL-60; neutrophil-like) cells were treated with the major DR risk factors viz. TNF-α (10ng/ml), IL-1β (10ng/ml), high glucose (HG, 30mM), and precursor of advanced glycation end product- methylglyoxal (MGO, 10μM), for 6 hours. dHL-60 activation was assessed by measuring superoxide level (using dihydroethidium dye, DHE; 5mM) and CD11b expression. The actin cytoskeleton was visualized in TRITC-Phalloidin-labeled cells using an epifluorescence microscope. Cytoskeletal parameters such as F-actin intensity and distribution, and morphological features (cell size and shape) were quantified using ImageJ.
Methods :
Differentiated HL-60 (dHL-60; neutrophil-like) cells were treated with the major DR risk factors viz. TNF-α (10ng/ml), IL-1β (10ng/ml), high glucose (HG, 30mM), and precursor of advanced glycation end product- methylglyoxal (MGO, 10μM), for 6 hours. dHL-60 activation assessed by measuring superoxide level using dihydroethidium (DHE;5mM) dye and CD11b expression. The actin cytoskeleton was visualized in TRITC-Phalloidin-labeled cells using an epifluorescence microscope. Cytoskeletal parameters such as F-actin intensity and distribution, and morphological features (cell size and shape) were quantified using ImageJ.
Results :
Our findings revealed that, among the aforementioned risk factors and when compared with untreated
dHL-60 cells, TNF-α elicits the highest increase in superoxide generation (1.58-fold; P<0.001) and CD11b expression (1.26-fold; P< 0.001). Further, neutrophil activation by TNF-α correlated with the highest increase (1.12-fold; p<0.01) in both cell size and F-actin polarization. In contrast, and rather unexpectedly, HG and MGO treatments caused a reduction in superoxide production, which was consistent with a concomitant reduction in cell size (12%; P<0.01) and F-actin polarization (30%; P<0.001).
Conclusions :
By correlating F-actin changes with superoxide generation, these findings raise the possibility of a potentially novel link between cytoskeletal dynamics and neutrophil activation in early DR. Ongoing studies are aimed at further elucidating the molecular mechanisms by which DR risk factors alter cytoskeletal dynamics in neutrophils and determining whether cytoskeletal modulation in neutrophils is necessary and sufficient to regulate its adhesion to and cytotoxicity toward retinal ECs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.