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 3

Salt does not induce an M1 phenotype in M(IL-4+IL-13)-activated macrophages.

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Salt does not induce an M1 phenotype in M(IL-4+IL-13)-activated macropha...
(A) BMDM were stimulated with LPS — M(LPS) — in the absence (none) or presence of an additional 40 mM NaCl or 80 mM mannitol for 24 hours. The expression of Nos2 was determined by qPCR. n = 6 (technical). *P < 0.001 vs. M(0); #P < 0.001 vs. M(LPS) none; and †P < 0.001 vs. M(LPS) + 80 mM mannitol. (B) Macrophages were activated to M(0), M(IL-4+IL-13), or M(LPS) as in A. Nos2, Il6, and Tnfa gene expression was determined by qPCR. n = 6 (technical). *P < 0.001 vs. M(0); #P < 0.05 vs. M(IL-4+IL-13) none; and †P < 0.05 vs. M(IL-4+IL-13) none and M(IL-4+IL-13) + 40 mM NaCl. (C) Macrophages were activated to M(0), M(IL-4+IL-13), or M(LPS) as in A, and genome-wide gene expression was determined by microarray. ChIP-seq was performed for H3K4me3 and H4ac chromatin modifications. Principle component (PC) analysis was performed on all genes in 2 independent, biological replicates. (D) Left: Macrophages were pretreated with an additional 40 mM NaCl and then stimulated with IL-4+IL-13 or LPS for 15, 30, or 60 min. The levels of phosphorylated and total IκBα were determined by Western blotting, with Hsp60 as control. Right: The expression of Ikba by qPCR. n = 6 (technical). *P < 0.01 vs. M(LPS). (E) CD38 surface expression as mean fluorescence intensity (MFI) by flow cytometry in macrophages activated to M(0), M(IL-4+IL-13), or M(LPS) alone (none) as in A. n = 2 technical replicates and the pooling of 3 independent experiments (total n = 6). *P < 0.01 vs. M(0) none, M(0) + 40 mM NaCl, M(IL-4+IL-13) none, and M(IL-4+IL-13) + 40 mM NaCl; and #P < 0.05 vs. M(LPS). (F) Left: The percentage of BMDM infected with Leishmania major after 72 hours. Right: Nitrate levels. The experiment was repeated independently 3 times and then pooled. n = 8–10 (technical). *P < 0.05 vs. M(0) none, M(0) + 40 mM NaCl, and M(IL-4+IL-13) none; **P < 0.01 vs. all other groups; #P < 0.0001 vs. M(IL-4+IL-13) + 40 mM NaCl; and †P < 0.001 vs. M(LPS) none. Statistics in A, B, and D–F were analyzed by 1-way ANOVA.