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 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 Article has an altmetric score of 66

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 9

High NaCl blunts the induction of AKT and mTOR signaling.

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High NaCl blunts the induction of AKT and mTOR signaling. (A) The effect...
(A) The effect of NaCl on AKT phosphorylation was determined by Western blotting. Macrophages were serum-starved overnight and then pretreated with 40 mM NaCl for 5 minutes, prior to stimulation with IL-4+IL-13 for 15, 30, or 60 minutes. The levels of total AKT were determined in a separate blot, with β-actin as a loading control. Also shown is phospho-p70S6K. Quantification of phospho-AKT/total AKT levels, normalized to t = 0, was obtained by pooling the mean levels from 3 independent experiments (total n = 4). *P < 0.05 vs. M(IL-4+IL-13) none by 2-way ANOVA. (B) Macrophages were stimulated with IL-4 and IL-13 in the absence (none) or with an additional 40 mM NaCl, or with an AKT inhibitor (LY294002, 50 μM) for 24 hours, and signature gene expression was analyzed by qPCR. n = 5 (technical). *P < 0.05 vs. M(0) none and M(0) + 40 mM NaCl; #P < 0.001 vs. M(IL-4+IL-13) none; †P < 0.001 vs. M(IL-4+IL-13) + 40 mM NaCl; and ‡P < 0.05 vs. M(IL-4+IL-13) + LY294002 none by 1-way ANOVA. (C) Schematic for the generation of BMDM with constitutively active Akt (NH[2]-terminally myristoylation signal-attached AKT; MyrAkt). Western blot (right) shows a robust induction of phosphorylated AKT after treatment of BMDM MyrAkt macrophages with TAT-Cre for 2 days. BMDM from WT macrophages treated with TAT-Cre similarly are shown. (D) BMDM from WT and MyrAkt mice were treated with TAT-Cre as in C and then stimulated with IL-4 and IL-13 in the absence (none) or with an additional 40 mM NaCl for 24 hours, and signature gene expression was analyzed by qPCR. Two independent stimulations were performed, which were then pooled (total n = 10–12 technical). *P < 0.05; **P < 0.01; and ***P < 0.001 by 2-way ANOVA.