Convenience versus Biological Significance: Are PMA-Differentiated THP-1 Cells a Reliable Substitute for Blood-Derived Macrophages When Studying in Vitro Polarization?

Serena Tedesco,Federica De Majo,Lucia Trevisi,Chiara Bolego,Peter W Zandstra,Andrea Cignarella,Jieun Kim,Gian Paolo Fadini,Annalisa Trenti,Libero Vitiello

doi:10.3389/fphar.2018.00071

Abstract

Human peripheral-blood monocytes are used as an established in vitro system for generating macrophages. For several reasons, monocytic cell lines such as THP-1 have been considered as a possible alternative. In view of their distinct developmental origins and phenotypic attributes, we set out to assess the extent to which human monocyte-derived macrophages (MDMs) and phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells were overlapping across a variety of responses to activating stimuli. Resting (M0) macrophages were polarized toward M1 or M2 phenotypes by 48-h incubation with LPS (1 μg/ml) and IFN-γ (10 ng/ml) or with IL-4 (20 ng/ml) and IL-13 (5 ng/ml), respectively. At the end of stimulation, MDMs displayed more pronounced changes in marker gene expression than THP-1. Upon assaying an array of 41 cytokines, chemokines and growth factors in conditioned media (CM) using the Luminex technology, secretion of 29 out of the 41 proteins was affected by polarized activation. While in 12 of them THP-1 and MDM showed comparable trends, for the remaining 17 proteins their responses to activating stimuli did markedly differ. Quantitative comparison for selected analytes confirmed this pattern. In terms of phenotypic activation markers, measured by flow cytometry, M1 response was similar but the established MDM M2 marker CD163 was undetectable in THP-1 cells. In a beads-based assay, MDM activation did not induce significant changes, whereas M2 activation of THP-1 decreased phagocytic activity compared to M0 and M1. In further biological activity tests, both MDM and THP-1 CM failed to affect proliferation of mouse myogenic progenitors, whereas they both reduced adipogenic differentiation of mouse fibro-adipogenic progenitor cells (M2 to a lesser extent than M1 and M0). Finally, migration of human umbilical vein endothelial cells was enhanced by CM irrespective of cell type and activation state except for M0 CM from MDMs. In summary, PMA-differentiated THP-1 macrophages did not entirely reproduce the response spectrum of primary MDMs to activating stimuli. We suggest that THP-1 be regarded as a simplified model of human macrophages when investigating relatively straightforward biological processes, such as polarization and its functional implications, but not as an alternative source in more comprehensive immunopharmacology and drug screening programs.

Highlights

Bone marrow–derived monocytes give rise to macrophages in some tissues, as well as during acute infection and inflammation (Geissmann et al, 2010; Hettinger et al, 2013; Wynn et al, 2013)
When it comes to investigating the pathways involved in macrophage activation and their pharmacological control, the source of macrophages, the definition of the activators, and the choice of surface markers and transcriptional regulation used to describe the type of activation are critical factors (Murray et al, 2014)
Both THP-1 and monocyte-derived macrophages (MDM) Conditioned Media (CM) were diluted to a twofold concentration compared to straight medium

Summary

INTRODUCTION

Bone marrow–derived monocytes give rise to macrophages in some tissues, as well as during acute infection and inflammation (Geissmann et al, 2010; Hettinger et al, 2013; Wynn et al, 2013). Macrophages are a heterogeneous and plastic cell population which, by integrating signals emanating from the environment, can be activated into a spectrum of phenotypes ranging from the pro-inflammatory classically activated (M1) to anti-inflammatory alternatively activated macrophages (M2) (Sica and Mantovani, 2012; Murray et al, 2014; Xue et al, 2014). The first two are main players in the repair and regeneration of skeletal muscle, a process in which macrophages play a paramount role (Juban and Chazaud, 2017); the third is instead involved in a wide range of physiological and pathological processes, from tissue repair to cancer growth in tight relationship with the inflammatory responses The output of these analyses may be relevant to cell model selection for several applications, such as immunopharmacology studies and drug screening programs

MATERIALS AND METHODS

RESULTS

DISCUSSION