Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.

Paola Italiani,Davide Lucchesi,Cristina Battaglia,Diana Boraschi,Silvio Bicciato,Emilia M C Mazza,Claudia Gemelli,Alexis Grande,Ingrid Cifola,Guido Poli

doi:10.1371/journal.pone.0087680

Abstract

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements.

Highlights

In the healthy organism the innate immune system provides the first line of defence against external or internal danger signals, and functions by triggering a protective inflammatory reaction develops through different phases, from initiation to full inflammation and destruction of the initiating agent, followed by resolution, and re-establishment of tissue integrity with restoration of the physiological tissue functions
This study aims at providing such characterisation by setting up a reliable and representative model, based on human primary monocytes, that allows us to accurately describing the development and regulation of human macrophage functions during the entire course of the inflammatory reaction
Transcriptomic analysis was performed on monocytes from each individual donor at five different stages of activation in comparison to control fresh monocytes: early inflammation (2–4 h), late inflammation (14 h); early and late resolution (24 and 48 h)

Summary

Introduction

In the healthy organism the innate immune system provides the first line of defence against external or internal danger signals, and functions by triggering a protective inflammatory reaction develops through different phases, from initiation to full inflammation and destruction of the initiating agent, followed by resolution, and re-establishment of tissue integrity with restoration of the physiological tissue functions. When in a tissue a damage or an infection takes place, the innate immune system is activated, setting in motion a local inflammatory response that includes the recruitment of leukocytes from blood (first neutrophils and them monocytes) and the production of a series of proinflammatory mediators, such as TNF-a and IL-1b, by local immune cells (in particular the resident macrophages). NK and T cells can enter the tissue in response to specific chemokines, and may influence the development of the inflammatory reaction by producing IFN-c, a potent monocyte/macrophage inflammatory activator. In the different microenvironment, innate immune cells produce a series of growth factors (including VEGF and TGF-b) thereby taking part in the final phase of tissue re-construction and re-establishment of homeostasis [1]

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