High salt reduces the activation of IL-4– and IL-13–stimulated macrophages
Katrina J. Binger, … , Jens Titze, Dominik N. Müller
Katrina J. Binger, … , Jens Titze, Dominik N. Müller
Published November 2, 2015; First published October 20, 2015
Citation Information: J Clin Invest. 2015;125(11):4223-4238. https://doi.org/10.1172/JCI80919.
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Research Article Cardiology Immunology Nephrology Vascular biology

High salt reduces the activation of IL-4– and IL-13–stimulated macrophages

Abstract

A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt–induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis.

Authors

Katrina J. Binger, Matthias Gebhardt, Matthias Heinig, Carola Rintisch, Agnes Schroeder, Wolfgang Neuhofer, Karl Hilgers, Arndt Manzel, Christian Schwartz, Markus Kleinewietfeld, Jakob Voelkl, Valentin Schatz, Ralf A. Linker, Florian Lang, David Voehringer, Mark D. Wright, Norbert Hubner, Ralf Dechend, Jonathan Jantsch, Jens Titze, Dominik N. Müller

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Figure 2

Effect of NaCl on M(IL-4+IL-13) activation is not related to changes in tonicity.

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Effect of NaCl on M(IL-4+IL-13) activation is not related to changes in ...
(A) Macrophages were stimulated with IL-4 and IL-13 in the absence (none) or with an additional 40 mM NaCl, or 80 mM urea or mannitol, as tonicity controls. Signature gene expression was analyzed by qPCR. The experiment was repeated at least 3 times independently. n = 6 (technical). *P < 0.0001 vs. M(0); #P < 0.05 vs. M(IL-4+IL-13) none and M(IL-4+IL-13) + mannitol; and †P < 0.01 vs. M(IL-4+IL-13) + urea by 1-way ANOVA. (B) Protein levels of ARG1 were determined by Western blotting after treatment, as in A. HSP60 loading control is also shown. Dot plots shows the quantification of the relative levels of ARG1 normalized to M(IL-4+IL-13) lysates, with n = 2–3 technical replicates and the pooling of 3 independent experiments (total n = 8). *P < 0.0001 (t test). (C) The effect of NaCl on the phosphorylation of STAT6 was determined by Western blotting. Macrophages were serum-starved overnight and then pretreated for 5 minutes with an additional 40 mM NaCl or 80 mM mannitol, prior to stimulation with IL-4+IL-13 for 15, 30, or 60 minutes. The levels of phosphorylated and total STAT6 were determined in separate blots, with β-actin used as loading control. The dot plot shows the quantification of relative levels of pSTAT6/STAT6 for each time point, normalized to t = 15 minutes of M(IL-4+IL-13) lysates, from the pooling of 2 independent experiments (n = 3 technical replicates).