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. 2017 Oct 24;21(4):1009-1020.
doi: 10.1016/j.celrep.2017.10.002.

Dendritic Cell Amiloride-Sensitive Channels Mediate Sodium-Induced Inflammation and Hypertension

Natalia R Barbaro  1 , Jason D Foss  1 , Dmytro O Kryshtal  1 , Nikita Tsyba  1 , Shivani Kumaresan  1 , Liang Xiao  1 , Raymond L Mernaugh  2 , Hana A Itani  1 , Roxana Loperena  3 , Wei Chen  1 , Sergey Dikalov  1 , Jens M Titze  1 , Bjorn C Knollmann  1 , David G Harrison  4 , Annet Kirabo  5
Affiliations

Dendritic Cell Amiloride-Sensitive Channels Mediate Sodium-Induced Inflammation and Hypertension

Natalia R Barbaro et al. Cell Rep. .

Abstract

Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox, and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin-1β (IL-1β) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation, and hypertension involving increased oxidative stress and IsoLG production in DCs.

Keywords: ENaC; NADPH oxidase; amiloride; calcium; dendritic cells; hypertension; isolevuglandins; oxidative stress; sodium chloride.

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Figures

Figure 1
Figure 1. High Salt Activates the NADPH Oxidase in Dendritic Cells Leading to Increased Superoxide Production through an Amiloride-Inhibitable Sodium Channel and Sodium-Calcium Exchanger
Mouse splenic dendritic cells were isolated and cultured for 24 hr in either normal-salt (150 mM NaCl) or high-salt (190 mM NaCl) media with or without the NADPH oxidase inhibitor, gp91dstat. Superoxide production was then measured by electron spin resonance. (A) Representative ESR signals showing the effect of high salt and gp91 ds-tat on dendritic cell superoxide production. (B) Average data showing the effect of high salt and gp91 ds-tat on dendritic cell superoxide production. (C) Flow cytometry representative histograms showing the effect of 15 min high-salt exposure on intracellular sodium. (D) Average data showing the effect of high salt on intracellular sodium. (E) qRT-PCR of sodium transporters: sodium-potassium-chloride cotransporter-1 (NKCC1), the sodium hydrogen exchangers (NHE1 and NHE6), the NCC, the sodium-calcium exchangers (NCX1 and NCX2), and the epithelial sodium channel (ENaC, α and γ subunits) on dendritic cells treated with normal or high salt. (F) Effect of sodium transporter inhibitors on phosphorylation of p47phox. (G) Western blot showing presence of the ENaC, α and γ subunits in dendritic cells treated with normal or high salt. (H) Effect of amiloride and benzamil on the high-salt-induced association of p47phox and gp91phox. (I) Effect of the Na+/H+ exchange inhibitor cariporide on the high-salt-induced association of p47phox and gp91phox. (J) Effect of α-ENaC targeted siRNA and non-targeting siRNA on expression of α-ENaC. (K) Effect of knockdown of α-ENaC on association of p47phox with gp91phox. (L) Effect of NHE1 targeted siRNA and non-targeting siRNA on expression of NHE1. (M) Effect of siRNA-mediated knockdown of NHE1 on the association of p47phox with gp91phox. (n = 5–6, *p < 0.05, **p < 0.01 versus normal-salt control, expressed as mean ± SEM).
Figure 2
Figure 2. Salt-Induced Activation of the NADPH Oxidase in Dendritic Cells Is Calcium- and PKC Dependent
Splenic dendritic cell lysates were immunoprecipitated for gp91phox, p47phox western blot was performed, the membranes were stripped, and gp91phox western blot was performed. (A) Effect of BAPTA-AM on the high-salt-induced association of p47phox with gp91phox. (B) Effect of the PKC inhibitor calphostin C on the high-salt-induced association of p47phox and gp91phox. (C) Representative tracing showing intracellular Ca2+ in a single dendritic cell in response to high salt using fluorescence photometry (IonOptix). (D) Average data showing the effect of the sodium calcium exchanger blocker, nickel chloride, on the high-salt-induced intracellular Ca2+ influx in dendritic cells. (E) Average data showing the effect of amiloride on the high-salt-induced intracellular Ca2+ influx in dendritic cells (n = 3–12, *p < 0.05 versus normal-salt control, expressed as mean ± SEM).
Figure 3
Figure 3. Excess Salt Induces Formation of Immunogenic IsoLGs and Expression of B7 Ligands in Dendritic Cells and Macrophages
Mouse splenocytes were cultured in normal-salt media (150 mM NaCl), high-salt media (190 mM NaCl), or normal-salt media with added mannitol (80 mM) as an osmotic control for 24 hr. (A) Flow cytometry gating strategy to identify dendritic cells and macrophages. (B and C) Flow cytometry representatives and average data showing intracellular staining for IsoLG-protein adducts in dendritic cells (B) and macrophages (C) using the single-chain antibody, D11 ScFv. (D and E) Flow cytometry representatives and average data showing surface expression of CD86 in dendritic cells (D) and macrophages (E). (F and G) Flow cytometry representatives and average data showing surface expression of CD80 in dendritic cells (F) and macrophages (G) (n = 5–6, *p < 0.05, **p < 0.01, ***p < 0.001 versus normal-salt control). (H) Representative flow cytometry histograms showing the effect of salt on dendritic cells lacking the NADPH oxidase subunit p22phox. (I) Average data showing the effect of salt on dendritic cells lacking the NADPH oxidase subunit p22phox (n = 6, **p < 0.01, expressed as mean ± SEM).
Figure 4
Figure 4. Dendritic Cells Exposed to High Salt Have Increased Production of IL-1b and IsoLG-Adducted Peptides in Their MHC
(A–C) Dendritic cells were isolated from spleens of mice, cultured in normal-salt or high-salt media and cytokines IL-1β (A), IL-6 (B), and TNF-α (C) were measured using a Luminex-based assay. (D) Effect of amiloride on the high-salt-induced production of IL-1β. (E) Surface peptides were eluted from dendritic cells treated with normal salt or high salt and dot blots were performed using the D-11 antibody. (F) Total amount of peptides eluted. (G) IsoLG-adducted peptides. (H) Flow cytometry representative histogram showing the effect of amiloride on the high-salt-induced accumulation of IsoLG-adducted peptides in dendritic cells (FMO control is shown). (I) Number of cells containing IsoLGs in normal-salt, high-salt, and high-salt + amiloride-treated dendritic cells. (J) Percentage of cells containing isolevuglandins in normal-salt, high-salt, and high-salt + amiloride-treated dendritic cells (n = 5–10, *p < 0.05 versus normal-salt control, expressed as mean ± SEM).
Figure 5
Figure 5. Dendritic Cells Exposed to High Salt Induce Production of Pro-hypertensive Cytokines by Primed T Cells
(A) Experimental strategy where dendritic cells were cultured in normal or high-salt media and then co-cultured with T cells isolated from mice that were exposed to repeated hypertensive challenges. (B) Flow cytometry gating strategy to identify T cells subsets. (C) Flow cytometry representatives showing intracellular staining for IFN-γ among CD8+ T cells in response to DCs treated with normal salt and high salt. (D) Average data showing the effect of high-salt-treated dendritic cells on IFN-γ production among CD8+ cells. (E) Flow cytometry representatives showing intracellular staining for IL-17 among CD8+ T cells in response to DCs treated with normal salt and high salt. (F) Average data showing the effect of high-salt-treated dendritic cells on IL-17 production among CD8+ cells. (G) Flow cytometry representatives showing intracellular staining for IFN-γ among CD4+ T cells in response to DCs treated with normal salt and high salt. (H) Average data showing the effect of high-salt-treated dendritic cells on IFN-γ production among CD4+ cells. (I) Flow cytometry representatives showing intracellular staining for IL-17 among CD4+ T cells in response to DCs treated with normal salt and high salt. (J) Average data showing the effect of high-salt-treated dendritic cells on IL-17 production among CD4+ cells. The Flow-minus-one (FMO) gating controls are shown in the top panels (n = 5–8, *p < 0.05 versus normal-salt control, expressed as mean ± SEM).
Figure 6
Figure 6. Salt-Activated Dendritic Cells Sensitize Mice to a Normally Suppressor Dose of Angiotensin II, Leading to Hypertension
(A) Dendritic cells were isolated from mouse spleens, cultured for 24 hr in normal salt, high salt, or high salt plus the isolevuglandin scavenger 2-HOBA and adoptively transferred into naive mice (106 DCs per mouse) via intravenous injection. Two weeks later, osmotic mini-pumps were implanted subcutaneously to deliver a low dose of angiotensin II (140 mg/kg/hr). (B–E) Systolic (B), diastolic (C), mean arterial blood pressure (D), and heart rate (E) were monitored using radio-telemetry (n = 5–6, *p < 0.05 versus normal-salt control, expressed as mean ± SEM).
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