Pro-inflammatory response of non-inflammatory Classical monocytes stimulated with LPSin vitro

Abstract

15 BACKGROUND: CD14 (Monocyte identifying Toll-Like Receptor) and CD16 (FcyRIII co-receptor, marker of 16 inflammatory monocytes) were used to define 3 subpopulations of circulating monocytes with different attributes in terms 17 of inflammatory and phagocytic capabilities. There are contradictory reports regarding response of circulating monocytes 18 to pro-inflammatory or non-inflammatory stimuli in vitro. Here we aimed to analyze the phenotypic changes in 19 circulating monocytes when stimulated with pro and non-inflammatory stimuli. 20 METHODS: Whole blood from 9 healthy donors was extracted and studied. Monocyte subpopulations were directly 21 measured using flow cytometry with PBMC Ficoll extraction method. Pro-inflammatory interleukin IL-1β was measured 22 by intracellular cytometry. Whole blood-extracted monocytes were stimulated using LPS and IL-4 as previously 23 described. Changes against non-stimulated (N-S) populations were statistically analyzed. 24 RESULTS: Compared to N-S, LPS-stimulated monocytes display a singular milieu of markers, with higher levels of 25 intracellular IL-1β in parallel raise of CD14+CD163-/CD14+CD163+ ratio. CD163 shows positive correlation with levels 26 of IL-1β. In t-SNE (T-distributed Stochastic Neighbor Embedding) analysis, after LPS stimulation, subpopulation 27 CD14+CD16-CD163-, containing mainly classical monocytes, show a higher number of IL-1β+ cells.28 CONCLUSION: Classical monocytes, the non-inflammatory subset, show higher levels of IL-1β in response to LPS 29 narrowing down to a new subpopulation of monocytes CD14+CD16-CD163-, which correlates better with this interleukin 30 response than widely used monocytes classification. Using CD163 in addition to CD16, we were able to show that 31 classical monocytes display the most intense response to LPS. Additionally, CD163 appears to be a suitable addition to 32 CD14-CD16 classification to improve its performance. 33

mTORC inhibitor Sirolimus deprograms monocytes in “cytokine storm” in SARS-CoV2 secondary hemophagocytic lymphohistiocytosis- like syndrome

“…We successfully classified M1 and M2 monocytes according to its membrane protein expression and intracellular interleukin production in healthy subjects. Human M1 monocytes in vitro were able to express higher levels of IL-6 and IL-1β after LPS stimulation [8] (Fig. 1).…”