Pro-inflammatory response of non-inflammatory Classical monocytes stimulated 1 with LPS in vitro. 2 Claudio Karsulovic 1,2 , Fabian Tempio 3,4 , Mercedes Lopez 3,4 , Julia Guerrero 1 , Annelise Goecke 1,2 3 1 Laboratorio de Inmunomodulación Neuroendocrina, Instituto de Ciencias Biomédicas, Facultad de 4 Medicina, Universidad de Chile, Santiago, Chile. 5 2 Sección de Reumatología, Hospital Clínico Universidad de Chile, Universidad de Chile, Santiago, 6 Chile. 7 3 Laboratorio de Regulación e Inmunología del Cáncer, Facultad de Medicina, Universidad de Chile, 8 Santiago, Chile. 9 4 Instituto Milenio de Inmunologia e Inmunoterapia, Facultad de Medicina, Universidad de Chile 10 * Correspondence: 11 Correspondence has to be addressed to Annelise Goecke at igoecke@hcuch.cl 12 Keywords: Monocytes, Phenotype, Classical, Intermediate, Non-Classical, LPS, flow 13 cytometry. 14 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 Introduction 34 In 2010, an international meeting sponsored by the IUIS (International Union of Immunological 35 Societies) and the WHO (World Health Organization), proposed a subset classification for circulating 36 monocytes (1). CD14 (Monocyte identifying Toll-Like Receptor) and CD16 (FcyRIII co-receptor, 37 marker of inflammatory monocyte) were used to define 3 subpopulations of circulating monocytes 38 with different attributes in terms of inflammatory protein expression and phagocytic capabilities (2). 39 Classical monocytes (CD14++CD16-) are 80 to 90% percent of the entire monocyte’s population, 40 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which this version posted May 25, 2020. ; https://doi.org/10.1101/2020.05.04.077537 doi: bioRxiv preprint