Circulating Neutrophils of Nonalcoholic Steatohepatitis Patients Show an Activated Phenotype and Suppress T Lymphocytes Activity
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 CD16HighCD62LHighCD11bHigh 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 CD16High CD62LDim CD11bHigh 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 CD16HighCD62LHighCD11bHigh 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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