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

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          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.

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          Most cited references63

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.
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              clusterProfiler: an R package for comparing biological themes among gene clusters.

              Increasing quantitative data generated from transcriptomics and proteomics require integrative strategies for analysis. Here, we present an R package, clusterProfiler that automates the process of biological-term classification and the enrichment analysis of gene clusters. The analysis module and visualization module were combined into a reusable workflow. Currently, clusterProfiler supports three species, including humans, mice, and yeast. Methods provided in this package can be easily extended to other species and ontologies. The clusterProfiler package is released under Artistic-2.0 License within Bioconductor project. The source code and vignette are freely available at http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                13 March 2023
                2023
                : 14
                : 1054588
                Affiliations
                [1] 1 Discipline of Surgery, Adelaide Medical School, The University of Adelaide , Adelaide, SA, Australia
                [2] 2 The Basil Hetzel Institute for Translational Health Research, The Queen Elizabeth Hospital , Adelaide, SA, Australia
                [3] 3 Department of Surgery-Otolaryngology Head and Neck Surgery, Central Adelaide Local Health Network , Adelaide, SA, Australia
                [4] 4 Medical Oncology, The Queen Elizabeth Hospital , Adelaide, SA, Australia
                [5] 5 Flinders Omics Facility, Department of Human Physiology, Flinders University , Adelaide, SA, Australia
                [6] 6 Discipline of Pharmacology, School of Biomedicine, The University of Adelaide , Adelaide, SA, Australia
                Author notes

                Edited by: Christine Gaboriaud, UMR5075 Institut de Biologie Structurale (IBS), France

                Reviewed by: Thierry Rabilloud, Chemistry and Biology of Metals, France; Jaewon Lim, Catholic University of Pusan, Republic of Korea

                *Correspondence: Kevin Fenix, kevin.fenix@ 123456adelaide.edu.au

                This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

                Article
                10.3389/fimmu.2023.1054588
                10040681
                36993962
                2b6ec45b-2a1a-494c-bde1-b5d758f516a9
                Copyright © 2023 Dhakal, Li, Ramezanpour, Houtak, Li, Bouras, Collela, Chegeni, Chataway, Drew, Sallustio, Vreugde, Smith, Maddern, Licari and Fenix

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 27 September 2022
                : 21 February 2023
                Page count
                Figures: 7, Tables: 1, Equations: 0, References: 63, Pages: 13, Words: 6529
                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.
                Categories
                Immunology
                Original Research

                Immunology
                m1 macrolphage,perhexiline,quantitative proteomics,thp-1 derived macrophages,macrophage polarisation

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