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. 2022 Sep 1;19(1):12.
doi: 10.1186/s12950-022-00309-8.

SILAC-based quantitative proteomics to investigate the eicosanoid associated inflammatory response in activated macrophages

Nicole Brace  1 , Ian L Megson  1 , Adriano G Rossi  2 , Mary K Doherty #  1 , Phillip D Whitfield #  3   4
Affiliations

SILAC-based quantitative proteomics to investigate the eicosanoid associated inflammatory response in activated macrophages

Nicole Brace et al. J Inflamm (Lond). .

Abstract

Background: Macrophages play a central role in inflammation by phagocytosing invading pathogens, apoptotic cells and debris, as well as mediating repair of tissues damaged by trauma. In order to do this, these dynamic cells generate a variety of inflammatory mediators including eicosanoids such as prostaglandins, leukotrienes and hydroxyeicosatraenoic acids (HETEs) that are formed through the cyclooxygenase, lipoxygenase and cytochrome P450 pathways. The ability to examine the effects of eicosanoid production at the protein level is therefore critical to understanding the mechanisms associated with macrophage activation.

Results: This study presents a stable isotope labelling with amino acids in cell culture (SILAC) -based proteomics strategy to quantify the changes in macrophage protein abundance following inflammatory stimulation with Kdo2-lipid A and ATP, with a focus on eicosanoid metabolism and regulation. Detailed gene ontology analysis, at the protein level, revealed several key pathways with a decrease in expression in response to macrophage activation, which included a promotion of macrophage polarisation and dynamic changes to energy requirements, transcription and translation. These findings suggest that, whilst there is evidence for the induction of a pro-inflammatory response in the form of prostaglandin secretion, there is also metabolic reprogramming along with a change in cell polarisation towards a reduced pro-inflammatory phenotype.

Conclusions: Advanced quantitative proteomics in conjunction with functional pathway network analysis is a useful tool to investigate the molecular pathways involved in inflammation.

Keywords: Gene ontology; Inflammation; Prostaglandins; Proteomic; RAW 264.7.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Secretion of inflammatory mediators. The log2-fold changes between PBS vehicle control and [Kdo2-lipid A + ATP] treated RAW264.7 cells in TNF (A) PGE2 (B), PGD2 (C) PGF2α (D) and PGJ2 (E) release are displayed. 100 ng/ml Kdo2 lipid A treatment commenced at t = 0, 2 mM ATP treatment began at t = 4 h. The mean of 3 separate experiments (in duplicate for TNF) are shown ± SD
Fig. 2
Fig. 2
Molecular, biological and cellular component ontology network. A The log2-fold changes (n = 3 ± SD) in protein abundance between PBS control and [Kdo2-lipid A + ATP] treated RAW264.7 cells following SILAC are presented. B The number of identified genes per term along with associated term p-value corrected with Bonferroni step down is shown. C The cellular components network of proteins with a significant fold change lower 0.5 (green) or higher than 2 (red) in relative abundance after stimulation with [Kdo2-lipid A + ATP] compared to a matched PBS control. Nodes (large circles) are linked by their common genes (red labels) based on a kappa score (≥ 0.4), with the label of the most significant terms displayed. Colour intensity of nodes represent significance of ontology. Gene fusion used
Fig. 3
Fig. 3
KEGG orthological network. A The log2-fold changes (n = 3 ± SD) in protein abundance between PBS control and [Kdo2-lipid A + ATP] treated RAW264.7 cells following SILAC are presented. B The number of identified genes per term, along with associated term p-value corrected with Bonferroni step down is shown. C The network of KEGG orthologies associated with the proteins with a significant fold change lower 0.5 (green) or higher than than 2 (red) in relative abundance after stimulation with [Kdo2-lipid A + ATP] compared to a matched PBS control. Nodes (large circles) are linked by their common genes (red labels) based on a kappa score (≥ 0.4), with the label of the most significant terms displayed. Colour intensity of nodes represent significance of ontology
Fig. 4
Fig. 4
Reactome Pathways network. A The log2-fold changes (n = 3 ± SD) in protein abundance between PBS control and [Kdo2-lipid A + ATP] treated RAW264.7 cells following SILAC are presented. B The number of identified genes per term along with associated term p-value corrected with Bonferroni step down is shown. C The network of Reactome pathways orthologies associated with the proteins with a significant fold change lower 0.5 (green) or higher than 2 (red) in relative abundance after stimulation with [Kdo2-lipid A + ATP] compared to a matched PBS control. Nodes (large circles) are linked by their common genes (red labels) based on a kappa score (≥ 0.4), with the label of the most significant terms displayed. Colour intensity of nodes represent significance of ontology
Fig. 5
Fig. 5
Reactome Reactions network. A The log2-fold changes (n = 3 ± SD) in protein abundance between PBS control and [Kdo2-lipid A + ATP] treated RAW264.7 cells following SILAC are presented. B The number of identified genes per term along with associated term p-value corrected with Bonferroni step down is shown. C The network of Reactome reactions orthologies associated with the proteins with a significant fold change lower 0.5 (green) or higher than 2 (red) in relative abundance after stimulation with [Kdo2-lipid A + ATP] compared to a matched PBS control. Nodes (large circles) are linked by their common genes (red labels) based on a kappa score (≥ 0.4), with the label of the most significant terms displayed. Colour intensity of nodes represent significance of ontology
Fig. 6
Fig. 6
Changes in Eicosanoid Related Proteins. The log2-fold changes in protein abundance between PBS control and [Kdo2-lipid A + ATP] treated RAW 264.7 cells are shown. Proteins COX5A, PTGR1 and HMGB1 were not present in vehicle control therefore log fold change cannot be obtained
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