Circulating Neutrophils of Nonalcoholic Steatohepatitis Patients Show an Activated Phenotype and Suppress T Lymphocytes Activity

Figure 3

PMNs activated phenotype of NASH patients. (a) Top panels, representative flow cytometry gating strategy (NASH patients) for identification ex vivo PMNs. FACS plots displaying, SSC-A versus FSC-A, singlet cells (FSC-H versus FSC-A), live cells (Zombie versus FSC-A), CD16 versus SSC-A, CD11b versus CD62L in ex vivo PMNs analysis. Bottom-left: histogram of mean fluorescence intensity (MFI) of CD62L and CD11b of ex vivo PMNs flow cytometric analysis obtained from healthy donors (), NAFL (), and NASH patients (). Bottom-right: representative histogram of flow cytometry analysis with an overlay showing CD62L and CD11b MFI in gated CD16^HighCD62L^HighCD11b^High of healthy donors (dashed line), NAFL (light gray), and NASH patients (dark gray). FMO control was represented by a gray histogram. Flow cytometry compensation beads positive control was represented by gray line. (b) PMNs obtained from healthy donors, NAFL, and NASH patients after 24 h of coculture with (PMNs+PBMCs) or without PBMCs (PMNs). Left panels: fold changes of CD62L (top) and CD11b (bottom) of MFI gated in CD16^High CD62L^Dim CD11b^High in healthy donors (), NAFL (), and NASH patients (). Fold change was calculated comparing PMNs with PBMCs respect to PMNs without PBMCs. Right panels: representative histogram with an overlay showing CD62L (top) and CD11b (bottom) MFI in gated CD16^HighCD62L^HighCD11b^High after 24 h of coculture with PBMC in healthy donors, NAFL, and NASH patients. Histogram with dashed line represents PMN, PMNs+PBMCs are represented by a dark line. FMO control was represented by gray histogram and Lipopolysaccharide (LPS) positive control was represented by a gray line. In all histograms reported, for each subgroup, the distribution of each subject/experiment is shown, and results are expressed as . Each dot represents one subject/experiment. Each subject has been studied in an independent experiment. In all experiments, statistics were performed by Mann-Whitney unpaired test, 2-tailed. value, , , ns: nonsignificant.

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