The immuno-metabolomics profile of cells presenting differential CCR5 phenotypes

Abstract

Background Chemokines are small molecules that form part of the cytokine family and direct the response of cells to chemical stimuli and thus serve as chemotactic factors. Cysteine-cysteine (C-C) chemokine receptor 5 (CCR5), expressed on the surface of various cells, is one of the most studied chemokine receptors. In addition to its more established role in immune function, particularly inflammation, CCR5, and its interaction with its ligand(s), also mediates various metabolic changes. Given the interplay between the immune and metabolic systems, CCR5 immune-directed changes should be measurable at a metabolic level also. However, the CCR5 gene, is prone to various mutations, some of which have an impact on the cell surface expression of the receptor and its associated functions. Although much has been done to characterise CCR5 structurally and from an immune perspective, CCR5-induced metabolic changes are less well described, and have been measured using more traditional analytical techniques, and in an inflamed/diseased model. Immuno-metabolism describes the interplay between the cells' immune and metabolic programs, and represents a novel approach to characterise CCR5 further, in terms of its role during on metabolism. Given the scarcity and difficulties associated with obtaining clinical samples, with varying levels of CCR5, two distinct cell lines with regulatable levels of CCR5 was sourced and their associated immuno-metabolomic profiles characterised. The Affinofile (derived from human embryonic kidney (HEK293) cells, also a control cell line in this study) and Cf2Th (derived from canine thymus cells) cells, were cultured in media containing non-toxic concentrations of antibiotics, to maintain their respective vectors. Treatment of the Affinofile and Cf2Th cells with Ponasterone A (Pon A) and doxycycline, respectively, proved non-toxic and induced differential levels of CCR5, as confirmed via flow cytometry. Analysis of the cell supernatant’s cytokine profile by flow cytometry revealed IL-2 as significantly different in cells with differential CCR5 levels as well as between the Affinofile and Cf2Th cell lines. This immunoregulatory cytokine is involved in cell growth, development, and homeostasis. The cells' intracellular metabolic profile as analysed using two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOFMS), indicated that those cells with higher levels of CCR5 showed metabolite markers indicative of upregulated energy metabolism and receptor stabilisation in line with the growth and metabolic demands of the receptor and its cell which supports the associated detection of IL-2. Investigating CCR5 in a non-activated/disease context informs the design of future experiments that may optimise CCR5-related immune and metabolic functions and its possible therapeutic roles.