Adiponectin Inhibits the Production of TNF-α, IL-6 and Chemokines by Human Lung Macrophages

Abstract

Background: Obesity is associated with an elevated risk of severe respiratory infections and inflammatory lung diseases. The objectives were to investigate 1) the production of adiponectin by human lung explants, 2) the expression of the adiponectin receptors AdipoR1 and AdipoR2 by human lung macrophages (LMs), and 3) the impact of recombinant human adiponectin and a small-molecule APN receptor agonist (AdipoRon) on LMs activation. Material and methods: Human parenchyma explants and LMs were isolated from patients operated for carcinoma. The LMs were cultured with recombinant adiponectin or AdipoRon and stimulated with lipopolysaccharide (10 ng ml^-1), poly (I:C) (10 µg ml^-1) or interleukin (IL)-4 (10 ng ml^-1) for 24 h. Cytokines or adiponectin, released by explants or LMs, were measured using ELISAs. The mRNA levels of AdipoR1 and AdipoR2 were determined using real-time quantitative PCR. AdipoRs expression was also assessed with confocal microscopy. Results: Adiponectin was released by lung explants at a level negatively correlated with the donor's body mass index. AdipoR1 and AdipoR2 were both expressed in LMs. Adiponectin (3-30 µg ml^-1) and AdipoRon (25-50 μM) markedly inhibited the LPS- and poly (I:C)-induced release of Tumor Necrosis Factor-α, IL-6 and chemokines (CCL3, CCL4, CCL5, CXCL1, CXCL8, CXCL10) and the IL-4-induced release of chemokines (CCL13, CCL17, CCL22) in a concentration-dependent manner. Recombinant adiponectin produced in mammalian cells (lacking low molecular weight isoforms) had no effects on LMs. Conclusion and implications: The low-molecular-weight isoforms of adiponectin and AdipoRon have an anti-inflammatory activity in the lung environment. Targeting adiponectin receptors may constitute a new means of controlling airways inflammation.

Keywords: LPS; Poly (I:C); adiponectin; adiporon; cytokine; human lung macrophage; obesity.

FIGURE 1

Experimental design.

FIGURE 2

APN release from lung explants. (A) Time-course of APN release from nonstimulated human lung explants cultured for 2, 4 and 24 h. Data are reported as the mean ± SEM of 5–11 independent experiments (***: p < 0.001). (B) Relationship between the APN production from unstimulated human lung explants after 24 h incubation assessed as the concentration in the supernantants and the donor’s BMI (n = 30). Spearman’s correlation coefficient r = -0.48; p = 0.008.

FIGURE 3

Expression of AdipoR1 and AdipoR2 transcripts by human LMs. Human LMs were cultured for 24 h in absence or presence of LPS (10 ng ml⁻¹), poly (I:C) (10 µg ml⁻¹) or IL-4 (10 ng ml⁻¹). AdipoR1 and AdipoR2 transcript levels were determined by RT-qPCR and normalized against those of a housekeeping gene (HPRT1). Data correspond to the mean ± SEM of 6 independent experiments (*: p < 0.05).

FIGURE 4

AdipoR1 and AdipoR2 are expressed on human LMs. AdipoR1 (A) and AdipoR2 (B) staining appears in green (AF488 fluorochrome). Immunostaining was evaluated using a SP5 Leica confocal microscopy (X63) and a spectral imaging acquisition.

FIGURE 5

Inhibitory effects of APN on the LPS- or poly(I:C)-induced cytokine release by human LMs. Macrophages were incubated with LPS (10 ng ml⁻¹, left column) or poly (I:C) (10 µg ml⁻¹, right column) in the absence or presence of APN (3, 10 or 30 µg ml⁻¹) for 24 h. Data are expressed as a percentage with respect to LPS or poly (I:C)-induced production. Results are shown as the mean ± SEM of 5–16 different experiments. Asterisks indicate significant effects of APN with respect to LPS or poly (I:C) alone (*:p < 0.05; **: p < 0.01; ***: p < 0.001).

FIGURE 6

Inhibitory effects of AdipoRon on the LPS- or poly(I:C)-induced cytokine release by human LMs. Macrophages were incubated with LPS (10 ng ml⁻¹, left column) or poly (I:C) (10 µg ml⁻¹, right column) in the absence or presence of AdipoRon (5, 10, 25, and 50 µM). Data are expressed as a percentage with respect to LPS or poly (I:C)-induced production. Results are shown as the mean ± SEM of 5–9 different experiments. Asterisks indicate significant effects of AdipoRon with respect to LPS or poly (I:C) alone (*:p < 0.05; **: p < 0.01; ***: p < 0.001).

FIGURE 7

Inhibitory effects of APN and AdipoRon on the IL-4-induced chemokine release by human LMs. Macrophages were incubated in the absence or presence of APN (3, 10 or 30 µg ml⁻¹) or AdipoRon (5, 10, 25 or 50 µM) before being stimulated with IL-4 (10 ng ml⁻¹) for 24 h. Data are expressed as a percentage with respect to IL-4-induced production. Results are shown as the mean ± SEM of 6–12 different experiments. Asterisks indicate significant effects with respect to IL-4 alone (*:p < 0.05; **: p < 0.01; ***: p < 0.001).