Salt Transiently Inhibits Mitochondrial Energetics in Mononuclear Phagocytes
BACKGROUND: Dietary high salt (HS) is a leading risk factor for mortality and morbidity. Serum sodium transiently increases postprandially but can also accumulate at sites of inflammation affecting differentiation and function of innate and adaptive immune cells. Here, we focus on how changes in extracellular sodium, mimicking alterations in the circulation and tissues, affect the early metabolic, transcriptional, and functional adaption of human and murine mononuclear phagocytes.METHODS: Using Seahorse technology, pulsed stable isotope-resolved metabolomics, and enzyme activity assays, we characterize the central carbon metabolism and mitochondrial function of human and murine mononuclear phagocytes under HS in vitro. HS as well as pharmacological uncoupling of the electron transport chain under normal salt is used to analyze mitochondrial function on immune cell activation and function (as determined by Escherichia coli killing and CD4(+) T cell migration capacity). In 2 independent clinical studies, we analyze the effect of a HS diet during 2 weeks (URL: http://www.clinicaltrials.gov.Unique identifier: NCT02509962) and short-term salt challenge by a single meal (URL: http://www.clinicaltrials.gov.Unique identifier: NCT04175249) on mitochondrial function of human monocytes in vivo.RESULTS: Extracellular sodium was taken up into the intracellular compartment, followed by the inhibition of mitochondrial respiration in murine and human macrophages. Mechanistically, HS reduces mitochondrial membrane potential, electron transport chain complex II activity, oxygen consumption, and ATP production independently of the polarization status of macrophages. Subsequently, cell activation is altered with improved bactericidal function in HS-treated M1-like macrophages and diminished CD4(+) T cell migration in HS-treated M2-like macrophages. Pharmacological uncoupling of the electron transport chain under normal salt phenocopies HS-induced transcriptional changes and bactericidal function of human and murine mononuclear phagocytes. Clinically, also in vivo, rise in plasma sodium concentration within the physiological range reversibly reduces mitochondrial function in human monocytes. In both a 14-day and single meal HS challenge, healthy volunteers displayed a plasma sodium increase of (x) over tilde = 2mM and (x) over tilde = 2.3mM, respectively, that correlated with decreased monocytic mitochondrial oxygen consumption.CONCLUSIONS: Our data identify the disturbance of mitochondrial respiration as the initial step by which HS mechanistically influences immune cell function. Although these functional changes might help to resolve bacterial infections, a shift toward proinflammation could accelerate inflammatory cardiovascular disease. ; S.G. was supported by the Bundesministerium für Bildung und Forschung funding MSTARS (Multimodal Clinical Mass Spectrometry to Target Treatment Resistance). D.N.M., H.B., N.W., and S.K.F. were supported by the Deutsche Forschungsgemeinschaft (German Research Foundation; Projektnummer 394046635 - SFB 1365). D.N.M. was supported by the Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK, 81Z0100106). J.J. received funding from the Deutsche Forschungsgemeinschaft (JA1993/6-1), Deutsche Forschungsgemeinschaft SFB 1350 grant (project No. 387509280, TPB5) and the Bavarian Ministry of Science and the Arts in the framework of the Bavarian Research Network "New Strategies Against Multi-Resistant Pathogens by Means of Digital Networking – bayresq.net." M.K. and N.W. were supported by the European Research Council under the European Union's Horizon 2020 research and innovation program (M.K.: 640116; N.W.: 852796). M.K. was further supported by a Strategic Action Plan for Limburg (Strategisch Actieplan voor Limburg in het Kwadraat, SALK) grant from the government of Flanders, Belgium, and by an Odysseus grant from the Research Foundation Flanders. N.W. is supported by a grant from the Corona-Stiftung. N.W. is participant in the Clinician Scientist Program funded by the Berlin Institute of Health. S.K. was supported by Impuls und Vernetzungsfond Aging and Metabolic Programming (AMPro, ZT-0026, Helmholtz Association). Acknowledgments The authors thank Jana Czychi, Gabriele N'diaye, Juliane Anders, Ute Gerhardt, May-Britt Köhler, and Fardad Ramezani for assistance. S.G. led and conceived the project, designed and performed most experiments, and analyzed and interpreted the data. H.B. conducted the clinical study, and performed PBMC isolation and flow-cytometric analysis together with S.G. P.N. performed murine BMDM and human peripheral blood monocyte bacterial killing and growth experiments. R.W., D.S., and A.G. performed analyses of ATP, tetramethylrhodamine ethyl ester, gene expression, and T cell migration in human macrophages. C.Z. together with S.G. measured and analyzed pulsed stable isotope-resolved metabolomics experiments. T.B. and E.T. performed sectioned transmission electron microscopy imaging and 3-dimensional reconstruction. V.M.P. performed mitochondrial isolation and ETC complex assays together with S.G. L.K. performed peripheral blood monocyte bacterial killing and cell culture experiments in human monocytes for intracellular Na+ measurement. M.V. performed intracellular Na+ quantifications. A. Maifeld, A. Mähler, and N.W. performed the clinical study, which was reanalyzed by S.G. and H.B. S.K.F. performed statistical analyses. K.B., J.S., and R.D. gave major conceptual input. D.N.M., S.K., J.J., and M.K. supervised the experiments and interpreted the data. S.G. and D.N.M. wrote the article with key editing by S.K., H.B., and M.K. and further input from all authors.