M-CSF- and L929-derived macrophages present distinct metabolic profiles with similar inflammatory outcomes

https://doi.org/10.1016/j.imbio.2020.151935Get rights and content

Abstract

Macrophages are essential components of the immune system. Macrophages can be derived from the bone marrow of mice with either recombinant M-CSF or L929 supernatant. Recent literature considers recombinant M-CSF- and L929-derived macrophages as equals, even though L929-derived macrophages are exposed to other substances secreted in the L929 supernatant, and not only M-CSF. Thus, we decided to perform a comparative analysis of both inflammatory and metabolic profiles of macrophages differentiated under the aforementioned conditions, which is relevant for standardization and interpretation of in vitro studies. We observed that, when treated with LPS, L929macs secrete lower levels of proinflammatory cytokines (TNF-α, IL-6, IL12) and present higher glycolysis and oxygen consumption when compared with M-CSFmacs. L929macs also have increased mitochondrial mass, with higher percentage of dysfunctional mitochondria. This sort of information can help direct further studies towards a more specific approach for macrophage generation.

Introduction

Macrophages are very plastic cells with distinct phenotypes. The classically activated macrophages (M1) and the alternatively activated macrophages (M2) are the best characterized populations (Jablonski et al., 2015; Castoldi et al., 2015). This division of M1 and M2 macrophages was proposed by Mills and collaborators from an analogy of the effector profile of these macrophages with specific T lymphocytes polarization and function (Mills et al., 2000). M1 macrophages are generated by lipopolysaccharide (LPS) and Th1 signals, such as IFN-γ (Castoldi et al., 2015). M1 macrophages present enhanced expression of inducible nitric oxide synthase (iNOS), and increased production of pro-inflammatory cytokines, such as IL-6, IL-1β, IL-12 and TNF-α (Diskin and Palsson-McDermott, 2018; Lumeng et al., 2007; Roszer, 2015; Pearce and Pearce, 2013). M2 macrophages are activated by Th2-type cytokines, such as IL-4 and IL-13 (Castoldi et al., 2015). M2 macrophages are often associated with anti-inflammatory functions and tissue repair, and are characterized by the expression of CD206, CD301, Arginase 1 and secretion of IL-10 (Lumeng et al., 2007; Roszer, 2015; Charles A Janeway et al., 2001; Galván-Peña and O’Neill, 2014). M2 macrophages are also metabolically distinct from M1 macrophages (Diskin and Palsson-McDermott, 2018; Breda et al., 2019; Correa da Silva et al., 2019). M2 macrophages rely on oxidative metabolism with increased mitochondrial respiration as the main source of adenosine triphosphate (ATP) (Pearce and Pearce, 2013; Van den Bossche et al., 2017; O’Neill et al., 2016). M1 macrophages are characterized by energetic shift to glycolysis, with increased lactate production (Pearce and Pearce, 2013; Van den Bossche et al., 2017; O’Neill et al., 2016).

Monocytes are circulating cells, which migrate to different tissues and can originate macrophages when stimulated with monocytic colony stimulating factor (M-CSF) (Weischenfeldt and Porse, 2008; Trouplin et al., 2013). In vitro, M-CSF is used to induce the differentiation of macrophages from bone marrow-derived precursors (Moraes-Vieira et al., 2014; Lacey et al., 2012; Lukic et al., 2017). The L929 cell line consists of immortalized fibroblasts, which are resident cells in the connective tissue of murine origin (Weischenfeldt and Porse, 2008; Trouplin et al., 2013) and can secrete high amounts of M-CSF. L929-derived supernatant is used for M-CSF-mediated macrophage differentiation (Weischenfeldt and Porse, 2008; Trouplin et al., 2013).

In 1991, Suresh and collaborators analysed the influence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) and conditioned medium of L929 in the production of cytokines from bone marrow derived macrophages (BMDMs). They observed that after stimulus with lipopolysaccharide (LPS), the production of IL-1 and TNF-α was increased in macrophages differentiated with GM-CSF compared with macrophages differentiated with conditioned medium of L929 (Suresh and Sodhi, 1991). On the other hand, albeit highly used, it is still not known whether recombinant M-CSF would promote the same pattern of response in comparison to L929-derived macrophages. Recent literature considers recombinant M-CSF- and L929-derived macrophages as equals, even though L929-derived macrophages are exposed to other substances secreted in the L929 supernatant, including small concentrations of GM-CSF, (Trouplin et al., 2013; Pang et al., 2000). Thus, we decided to perform a comparative analysis of both the inflammatory and metabolic profiles of BMDMs differentiated under the aforementioned conditions, which is relevant for standardization and interpretation of in vitro studies.

Section snippets

Macrophage differentiation and polarization

Macrophages were derived from the bone marrow of C57BL/J6 mice as described previously (Moraes-Vieira et al., 2016a, b). We collected cells from the femur and tibia and plated them in complete RPMI media (10 % FBS, 1 % Penicillin-Streptomycin, 1 % Non-Essential Amino acids and 1% MEM Vitamin, 1 % sodium pyruvate – Gibco) containing either 10 % of L929 supernatant (which was the optimal concentration for macrophage differentiation – Supplementary Fig. 1A and B) or 20 ng/mL recombinant M-CSF for

L929-derived macrophages produce less pro-inflammatory cytokines than M-CSF-derived macrophages but maintain microbicidal efficiency

We first evaluated the cytokine profile of macrophages differentiated with recombinant M-CSF (M-CSFmacs) or L929 supernatant (L929macs) for 6 days and activated with LPS (Fig. 1A). LPS-activated L929macs displayed similar gene expression of Tnfα, Il12 and Il10 but increased Il6 and Nos2 expression compared to M-CSFmacs (Fig. 1B). However, LPS-activated L929macs secreted higher levels of IL-10 and reduced levels of TNF-α and IL-12, while IL-6 levels remained similar (Fig.1C). Our results show

Discussion

In the present work we explored the metabolic and inflammatory differences between macrophages polarized using commercial M-CSF and the supernatant of L929. Our main findings are: (i) L929macs produced less inflammatory cytokines than M-CSFmacs; (ii) both macrophages are dependent on glycolysis and L929macs are more metabolically active; (iii) L929 M1 secrete reduced levels of pro-inflammatory cytokines than M-CSF M1 macrophages.

Although others have shown that L929macs generate less

Authors contribution

Lauar Monteiro and Gustavo G. Davanzo designed and performed experiments, analyzed data, wrote the manuscript and made the figures. Felipe Corrêa da Silva, Cristhiane Fávero de Aguiar, Jessica Rodrigues de Andrade, Ana Codo, Jessica Pereira and Leonardo Pimentel de Freitas performed experiments. P. M. M. Moraes-Vieira designed, wrote and edited manuscript.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

We thank the São Paulo Research Foundation - FAPESP (grant numbers 2015/15626-8, 2016/23328-0, 2016/18031-8, 2019/06372-3), the Brazilian National Council for Scientific and Technological Development - CNPq, and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Code 001.Graphical abstract was created with Biorender - biorender.com.

References (33)

  • J. Van den Bossche et al.

    Macrophage Immunometabolism: Where Are We (Going)?

    Trends Immunol.

    (2017)
  • C. Aguer et al.

    Galactose enhances oxidative metabolism and reveals mitochondrial dysfunction in human primary muscle cells

    PLoS One

    (2011)
  • A. Castoldi et al.

    The macrophage switch in obesity development

    Front. Immunol.

    (2015)
  • J. Charles A Janeway et al.

    Immunobiology

    (2001)
  • F. Correa-da-Silva et al.

    Mitoimmunity-when mitochondria dictates macrophage function

    Cell Biol. Int.

    (2018)
  • C. Diskin et al.

    Metabolic modulation in macrophage effector function

    Front. Immunol.

    (2018)
  • Cited by (9)

    View all citing articles on Scopus
    View full text