Figure 6. Disulfide-HMGB1+IRI+ patient LF activates monocytes to translocate nuclear HMGB1 into cytoplasmic vesicles and become more pro-inflammatory.

PBMC-derived monocytes from healthy, third-party donors were cultured with patient LF (n=6; 3 IRI-, 3 IRI+) for 2 hours to determine HMGB1 localization by confocal microscopy or 3 days to determine functional phenotype by flow cytometry. (A) Representative images of monocytes stained by multicolor IF for CD68 (red), HMGB1 (pink), DAPI (blue), and LAMP1 (green), and z-stack images were acquired by confocal microscopy and reconstructed in Imaris. Scale bars = 2 um (B) Quantification of monocyte-specific cytoplasmic HMGB1 in LAMP1+ vesicles from reconstructed confocal laser-scanning microscopy Z-stacks. Data are presented as dots for each of three separate experiments with long lines indicating mean. (C) Stimulated monocytes were multiplex stained with a panel of directly-labeled antibodies and run on a flow cytometer to determine surface expression levels of CD14, CD16, CD11b, CD68, HLA-DR, CD80, CD86, CD66a, Gal-9, TIM-3, TIM-4, and PD-L1. Geometric mean fluorescence intensity (GeoMFI) for the respective surface marker on cells stimulated by n=77; 43 IRI- versus 34 IRI- LF are shown. Data are presented as Tukey box-and-whisker plots: whiskers are inner fences reaching 1.5 times the interquartile range and boxes represent the interquartile ranges, dots indicate outlying values and lines represent median values. ns = no significance; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by Student’s T test.