Mitochondrial Ultrastructure and Activity Are Differentially Regulated by Glycolysis-, Krebs Cycle-, and Microbiota-Derived Metabolites in Monocytes.

Several intermediate metabolites harbour cell-signalling properties, thus, it is likely that specific metabolites enable the communication between neighbouring cells, as well as between host cells with the microbiota, pathogens, and tumour cells. Mitochondria, a source of intermediate metabolites, participate in a wide array of biological processes beyond that of ATP production, such as intracellular calcium homeostasis, cell signalling, apoptosis, regulation of immune responses, and host cell-microbiota crosstalk. In this regard, mitochondria's plasticity allows them to adapt their bioenergetics status to intra- and extra-cellular cues, and the mechanisms driving such plasticity are currently a matter of intensive research. Here, we addressed whether mitochondrial ultrastructure and activity are differentially shaped when human monocytes are exposed to an exogenous source of lactate (derived from glycolysis), succinate, and fumarate (Krebs cycle metabolic intermediates), or butyrate and acetate (short-chain fatty acids produced by intestinal microbiota). It has previously been shown that fumarate induces mitochondrial fusion, increases the mitochondrial membrane potential (Δψm), and reshapes the mitochondrial cristae ultrastructure. Here, we provide evidence that, in contrast to fumarate, lactate, succinate, and butyrate induce mitochondrial fission, while acetate induces mitochondrial swelling. These traits, along with mitochondrial calcium influx kinetics and glycolytic vs. mitochondrial ATP-production rates, suggest that these metabolites differentially shape mitochondrial function, paving the way for the understanding of metabolite-induced metabolic reprogramming of monocytes and its possible use for immune-response intervention.

A flow-cytometry method for analyzing the composition of membrane rafts.

Membrane rafts are involved in a broad variety of biological processes. Their protein composition under growth factor stimulation, anti-inflammatory or proinflammatory microenvironments, or in the course of pathogenic infections still remains to be determined. However, current techniques aimed at the identification of particular proteins on membrane rafts are not devoid of pitfalls. Membrane rafts were obtained by detergent-free based differential centrifugation from Jurkat T cells and J774 macrophages. Membrane rafts were labeled with fluorochrome-labeled antibodies directed against different cell membrane molecules, and with fluorochrome-labeled cholera toxin B that targets GM1 and analyzed by flow cytometry. CD3, CD11a, and GM1 were shown to be differentially expressed on Jurkat T cell-derived membrane rafts, indicating heterogeneity in membrane rafts composition. On the other hand, it was shown in J774 cell-derived membrane rafts that most but not all CD14 is present in the GM1-containing membrane fragments, thus confirming the heterogeneity of membrane rafts composition in other cell lines. The method described here allows the fluorometric assessment of the relative expression of more than one membrane raft component at a time, and at a single vesicle level in a fast and sensitive manner. This method seems to be a suitable approach to evaluate the molecular heterogeneity in membrane rafts composition.

Expression of GM1, a marker of lipid rafts, defines two subsets of human monocytes with differential endocytic capacity and lipopolysaccharide responsiveness.

Monocytes constitute 5-10% of total human peripheral blood leucocytes and remain in circulation for several days before replenishing the tissue macrophage populations. Monocytes display heterogeneity in size, granularity and nuclear morphology, and in the expression of cell membrane molecules, such as CD14, CD16, CD32, CD64, major histocompatibility complex class II, CCR2, CCR5, among others. This has led to the suggestion that individual monocyte/macrophage populations have specialized functions within their microenvironments. This study provides evidence for the occurrence of two peripheral blood monocyte subpopulations on the basis of their differential expression of GM1, a sphingolipid found mostly in lipid rafts, a CD14(+) GM1(low) population and a CD14(+) GM1(high) population comprising about 97.5% and 2.5% of total CD14(+) cells, respectively. GM1 expression correlates with functional differences in terms of endocytic activity, susceptibility to mycobacterial infection, and response to lipopolysaccharide (LPS) (modulation of Toll-like receptor-4 expression). CD14(+) GM1(low) cells proved to be less endocytic and more responsive to LPS, whereas CD14(+) GM1(high) cells are more endocytic and less responsive to LPS. In addition, during monocyte to macrophage differentiation in vitro, the percentage of CD14(+) GM1(high) cells increases from about 2.5% at day 1 to more than 50% at day 7 of culture. These results suggest that GM1(low) and GM1(high) monocytes in peripheral blood, represent either different stages of maturation or different subsets with specialized activities. The expression of CD16 on GM1(high) favours the first possibility and, on the other hand that up-regulation of GM1 expression and probably lipid rafts function is involved in the monocyte to macrophage differentiation process.