Classical monocytes, the non-inflammatory phenotype, display the most robust pro-inflammatory response to LPS in vitro after immediate cytometric analysis

BACKGROUNDCD14 (Monocyte identifying Toll-Like Receptor) and CD16 (FcyRIII co-receptor, marker of inflammatory monocytes) were used to define 3 subpopulations of circulating monocytes with different attributes in terms of inflammatory and phagocytic capabilities. There are contradictory reports regarding response of circulating monocytes to pro-inflammatory or non-inflammatory stimuli in vitro. Here we aimed to analyze the phenotypic changes in circulating monocytes when stimulated with pro and non-inflammatory stimuli. METHODSWhole blood from 9 healthy donors was extracted and studied. Monocyte subpopulations were directly measured using flow cytometry with PBMC Ficoll extraction method. Pro-inflammatory interleukin IL-1{beta} was measured by intracellular cytometry. Whole blood-extracted monocytes were stimulated using LPS and IL-4 as previously described. Changes against non-stimulated (N-S) populations were statistically analyzed. RESULTSCompared to N-S, LPS-stimulated monocytes display a singular milieu of markers, with higher levels of intracellular IL-1{beta} in parallel raise of CD14+CD163-/CD14+CD163+ ratio. CD163 shows positive correlation with levels of IL-1{beta}. In t-SNE (T-distributed Stochastic Neighbor Embedding) analysis, after LPS stimulation, subpopulation CD14+CD16-CD163-, containing mainly classical monocytes, show a higher number of IL-1{beta}+ cells. CONCLUSIONClassical monocytes, the non-inflammatory subset, show higher levels of IL-1{beta} in response to LPS narrowing down to a new subpopulation of monocytes CD14+CD16-CD163-, which correlates better with this interleukin response than widely used monocytes classification. Using CD163 in addition to CD16, we were able to show that classical monocytes display the most intense response to LPS. Additionally, CD163 appears to be a suitable addition to CD14-CD16 classification to improve its performance.