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

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 8

Salt modulates M(IL-4+IL-13) cellular metabolism.

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Salt modulates M(IL-4+IL-13) cellular metabolism. (A) Macrophages were a...
(A) Macrophages were activated to M(IL-4+IL-13) ± high salt for 24 hours as before, and gene expression was analyzed by microarray. M(IL-4+IL-13) signature genes from Figure 1 and Slc6a12 are indicated. Salt-modulated M(IL-4+IL-13) genes were subjected to GO analysis (right). The top 20 GO terms (FDR < 0.065) are shown. (B) Schematic of the major cellular metabolic pathways: glycolysis and OXPHOS. (C) OCR of unstimulated (M[0]) or M(IL-4+IL-13)-activated macrophages without (none) or with high salt for 24 hours, followed by sequential treatment with Oligomycin (to inhibit mitochondrial respiration), FCCP (to elucidate maximal respiration), and rotenone plus antimycin A (Rot+A) (to measure nonmitochondrial respiration). Also basal OCR, basal ECAR, and SRC in 3 independent experiments were pooled (total n = 14). *P < 0.05 vs. M(0) and #P < 0.05 vs. M(IL-4+IL-13). (D) Mitochondrial mass by staining cells with MitoTracker red and flow cytometry. n = 6 (2 independent experiments pooled). (E) Mitochondrial content was measured by the ratio of mitochondrial (mt) DNA to nuclear (N) DNA. n = 5 (technical). (F) ECAR of M(0) or M(IL-4+IL-13) activated as in C, followed by sequential treatment with glucose (to initiate glycolysis), Oligomycin, and 2-deoxy glucose (DG) (to inhibit glycolysis). The rate of glycolysis was calculated based on the difference between basal ECAR and the maximal ECAR, following glucose stimulation. Two independent experiments were pooled (total n = 10). *P < 0.05 vs. M(0) and #P < 0.05 vs. M(IL-4+IL-13). (G) One-hour lactate production from supernatants by fluorometric assay. Two independent experiments were pooled (total n = 7). (H) Glucose uptake of macrophages activated as indicated for 24 hours, followed by incubation of the cells with 2NB-DG for 1 hour and analysis by flow cytometry. Three independent experiments were pooled (total n = 9). *P < 0.05 vs. M(0) none. Statistics in C, F, and H) were analyzed by 1-way ANOVA.