Expression of cell-surface activation markers on human CD15⁺ cells after selected non esterified fatty acid supplementations

Abstract

Background: Cell surface marker (CD) expressions and membrane lipid changes characterise circulating neutrophil transmigration into inflammation sites. Specific CD expressions mandate neutrophil activation. Lipid changes alter membrane fluidity, deformability, cell spreading and transmigration. Circulating neutrophil, predominantly CD15 positive (CD15⁺), recruitment to the lungs and activation is a crucial innate immune response in Acute Respiratory Distress Syndrome (ARDS) pathology. During transmigration, their membrane lipids rapidly acquire significant whole cell arachidonic acid enrichment. The source of arachidonate is unclear but neutrophils incorporate non-esterified fatty acids (NEFA) from their microenvironment, adjusting cellular fatty acyl-CoA pools. We hypothesised that inflammation-associated, specific NEFA incorporation(s) into neutrophil membrane lipids mandate activation. We evaluated patterns of synthesis, composition and turnover of phosphatidylcholine (PC), alongside transmigration markers, following NEFA supplementations of freshly isolated human CD15⁺ cells. 

Methods: CD15⁺ cells were collected from venous blood samples of 5 healthy volunteers using the autoMACS system with CD15⁺ microbeads. Isolated CD15⁺ cells were incubated for 3 h with methyl-D₉-choline chloride. NEFA supplements were added separately or in combination. Modified Bligh and Dyer lipid extracts were analysed by mass spectrometry. Unlabelled PC composition was determined by precursor scans of the m/z + 184 and the deuteriated m/z + 193 fragment was used to report newly synthesised methyl-D₉-choline labelled PC. 

Results: Four NEFAs were chosen, oleic acid, linoleic acid, palmitic acid and arachidonic acid. All supplementations downregulated CD62L expression and increased expressions of CD11a and CD11b, hallmarks of activation. Membrane PC composition consisted of di-acyl species (59%) and alkylacyl PC species (41%), primary PC being PC34:1. Incorporation of methyl-D₉-into polyunsaturated PC species was consistently elevated compared with endogenous composition. NEFA supplementations did not change bulk endogenous PC composition. There was lower fractional methyl-D₉ enrichment with oleic acid compared to other NEFAs and the fractional enrichment varied between individual species. Conclusions: CD15⁺ microenvironmental exposure to every NEFA investigated resulted in expression of cellular activation markers. NEFA-defined changes in synthesis patterns of individual molecular species of membrane PC were consistent with membrane fluidity changes facilitating recruitment of activation markers and transmigration. Data suggest that altered neutrophil exposure to NEFA in vivo may potentially regulate their immune response.

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