Hypertonic saline induces inflammation in human macrophages through the NLRP1 inflammasome

Abstract

Nebulized hypertonic saline (3-7%) is commonly used to increase mucociliary clearance in patients with chronic airway disease and/or virus infections. However, altered salt concentrations may contribute to inflammatory responses. The aim of this study was to investigate whether 500 mM NaCl (3%) triggers inflammation in human macrophages and identify the molecular mechanisms involved. NaCl-induced pyroptosis, IL-1β, IL-18 and ASC speck release were measured in primary human monocyte-derived macrophages. Treatment with the recombinant IL-1 receptor antagonist anakinra or the NLRP3 inhibitor MCC950 did not affect NaCl-mediated inflammasome assembly. Knock-down of NLRP1 expression, but not of NLRP3 and NLRC4, reduced NaCl-induced pyroptosis, pro-inflammatory cytokine and ASC speck release from human THP-1-derived macrophages. Data from this study suggest that 3% NaCl-induced inflammatory responses in human macrophages depend on NLRP1 and inflammasome assembly. Targeting inflammation in addition to inhalation with hypertonic saline may benefit patients with inflammatory airway disease.

Fig. 1. NaCl alone or with LPS induces inflammatory cell death, IL-1β and IL-18 release in primary human monocyte-derived macrophages (MDM).

A Inflammatory cell death evaluated through LDH assay after 8 h of stimulation; IL-1β (B) and IL-18 (C) release measured through MSD assays. LPS lipopolysaccharide, Unstim Unstimulated cells. N > 9; Kruskal–Wallis test. *: p < 0.05; ***: p < 0.001; ****: p < 0.0001.

Fig. 2. Anakinra and MCC950 do not alter NaCl-induced inflammation in primary human MDM.

Supernatants were analysed after 8 h of stimulation. A, D Inflammatory cell death evaluated through LDH assays; B, E IL-1β concentrations measured through MSD assays; C, F IL-18 concentrations measured through MSD assays. N ≥ 4; Kruskal–Wallis. *: p < 0.05; *: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Fig. 3. The stimulation with NaCl induces the release of ASC specks in primary human MDM.

A ASC specks in cell culture supernatants following 8 h of stimulation with LPS, NaCl, or NaCl and LPS, were quantified via flow cytometry and normalised to the unstimulated controls; B MCC950, but not Anakinra, reverts NaCl effect, reducing the amount of ASC specks, in a biologically meaningful manner; data were normalised to the relative unstimulated control. N > 4; Kruskal–Wallis test. ***: p < 0.001; ****: p < 0.0001.

Fig. 4. Hypertonic saline induces inflammatory cell death and IL-1β release in THP-1 derived macrophages.

Inflammatory cell death, IL-1β and IL-18 release were evaluated after 8 h of stimulation. A Cell death measured via LDH assay; data were normalised to the unstimulated controls; B, C IL-1β and IL-18 concentrations respectively measured through MSD assays; N ≥ 7; statistical analysis performed with Mann–Whitney test. *: p < 0.05; ****: p < 0.0001.

Fig. 5. Anakinra and MCC950 do not alter NaCl-induced inflammation in THP-1 derived macrophages.

Supernatants were analysed after 8 h of stimulation. A, D Inflammatory cell death evaluated through LDH assays; B, E IL-1β concentration measured through MSD assays; C, F IL-18 concentrations measured through MSD assays; G ASC specks in cell supernatants following 8 h of stimulation with NaCl, or NaCl and LPS, were quantified via flow cytometry and normalised to unstimulated controls. Data were normalised to the relative unstimulated control. N ≥ 4; Kruskal–Wallis. ***: p < 0.001; ****: p < 0.0001.

Fig. 6. NLRP1 knock-down inhibits NaCl-induced release of IL-1β, IL-18, and ASC specks.

A Inflammatory cell death evaluated through LDH assays; B, C IL-1β and IL-18 measured through MSD assays; D Extracellular ASC specks quantified via flow cytometry and normalised to the unstimulated controls. Statistical significance was calculated using the Mann–Whitney test; N ≥ 4; *: p ≤ 0.05.