Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation

Bimala Dhakal; Celine Man Ying Li; Mahnaz Ramezanpour; Ghais Houtak; Runhao Li; George Bouras; Alex Collela; Nusha Chegeni; Tim Kennion Chataway; Paul Drew; Benedetta C Sallustio; Sarah Vreugde; Eric Smith; Guy Maddern; Giovanni Licari; Kevin Fenix

doi:10.3389/fimmu.2023.1054588

Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation

Front Immunol. 2023 Mar 13:14:1054588. doi: 10.3389/fimmu.2023.1054588. eCollection 2023.

Authors

Bimala Dhakal^{1

2}, Celine Man Ying Li^{1

2}, Mahnaz Ramezanpour^{1

2

3}, Ghais Houtak^{1

2

3}, Runhao Li^{1

2

4}, George Bouras^{1

2

3}, Alex Collela⁵, Nusha Chegeni⁵, Tim Kennion Chataway⁵, Paul Drew^{1

2}, Benedetta C Sallustio^{2

6}, Sarah Vreugde^{1

2

3}, Eric Smith^{1

2

4}, Guy Maddern^{1

2}, Giovanni Licari^{2

6}, Kevin Fenix^{1

2

3}

Affiliations

¹ Discipline of Surgery, Adelaide Medical School, The University of Adelaide, Adelaide, SA, Australia.
² The Basil Hetzel Institute for Translational Health Research, The Queen Elizabeth Hospital, Adelaide, SA, Australia.
³ Department of Surgery-Otolaryngology Head and Neck Surgery, Central Adelaide Local Health Network, Adelaide, SA, Australia.
⁴ Medical Oncology, The Queen Elizabeth Hospital, Adelaide, SA, Australia.
⁵ Flinders Omics Facility, Department of Human Physiology, Flinders University, Adelaide, SA, Australia.
⁶ Discipline of Pharmacology, School of Biomedicine, The University of Adelaide, Adelaide, SA, Australia.

Abstract

Background: Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced.

Methods: We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS).

Results: PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1β secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways.

Conclusion: This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

Keywords: M1 macrolphage; THP-1 derived macrophages; macrophage polarisation; perhexiline; quantitative proteomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Differentiation
Humans
Inflammation / metabolism
Macrophages
Perhexiline* / metabolism
Perhexiline* / pharmacology
Proteome / metabolism
Proteomics*

Substances

Perhexiline
Proteome

Grants and funding

This work was supported by a The Hospital Research Foundation (THRF)/Cancer Council SA Beat Cancer Hospital Research Package (GM). BD was supported by a Schlumberger Foundation Faculty for Future Fellowship, and KF was supported by a THRF Early Career Fellowship.