Regulation of YKL-40 expression by corticosteroids: effect on pro-inflammatory macrophages in vitro and its modulation in COPD in vivo

Abstract

Background: Macrophages constitute a heterogeneous cell population with pro- (MΦ1) and anti-inflammatory (MΦ2) cells. The soluble chitinase-like-protein YKL-40 is expressed in macrophages and various other cell types, and has been linked to a variety of inflammatory diseases, including COPD. Dexamethasone strongly reduces YKL-40 expression in peripheral blood mononuclear cells (PBMC) in vitro. We hypothesized that: a) YKL-40 is differentially expressed by MΦ1 and MΦ2, b) is decreased by corticosteroids and c) that long-term treatment with inhaled corticosteroids (ICS) affects YKL-40 levels in serum and sputum of COPD patients.

Methods: Monocytes of healthy subjects were cultured in vitro for 7 days with either GM-CSF or M-CSF (for MΦ1 and MΦ2, respectively) and stimulated for 24 h with LPS, TNFα, or oncostatin M (OSM). MΦ1 and MΦ2 differentiation was assessed by measuring secretion of IL-12p40 and IL-10, respectively. YKL-40 expression in macrophages was measured by quantitative RT-PCR (qPCR) and ELISA; serum and sputum YKL-40 levels were analyzed by ELISA.

Results: Pro-inflammatory MΦ1 cells secreted significantly more YKL-40 than MΦ2, which was independent of stimulation with LPS, TNFα or OSM (p < 0.001) and confirmed by qPCR. Dexamethasone dose-dependently and significantly inhibited YKL-40 protein and mRNA levels in MΦ1. Serum YKL-40 levels of COPD patients were significantly higher than sputum YKL-40 levels but were not significantly changed by ICS treatment.

Conclusions: YKL-40 secretion from MΦ1 cells is higher than from MΦ2 cells and is unaffected by further stimulation with pro-inflammatory agents. Furthermore, YKL-40 release from cultured monocyte-derived macrophages is inhibited by dexamethasone especially in MΦ1, but ICS treatment did not change YKL-40 serum and sputum levels in COPD. These results indicate that YKL-40 expression could be used as a marker for MΦ1 macrophages in vitro, but not for monitoring the effect of ICS in COPD.

Trial registration: ClinicalTrials.gov, registration number: NCT00158847.

Figures

Fig. 1

Characterization of differentiated MΦ1 and MΦ2. a: IL-12p40 (left panel) and IL-10 (right panel) in supernatants of MΦ1 and MΦ2, respectively, stimulated with LPS for 24 h. Data represent means with SEM of 6 donors, ** p < 0.01 or *** p < 0.001 between MΦ1 and MΦ2. b: Flow cytometry analysis for CD68, CD14 and CD163 (left, middle and right panel, respectively) for MΦ1 and MΦ2 (upper and lower panel, respectively)

Fig. 2

MΦ1 produce more YKL-40 compared to MΦ2. a: YKL-40 secretion measured by ELISA (left panel, 6 donors) and mRNA levels (normalized expression, right panel, 5 donors) in MΦ1 and MΦ2 after medium and LPS stimulation for 24 h. * p < 0.05 or *** p < 0.001 between MΦ1 and MΦ2. b: Flow cytometry analysis of MΦ1 and MΦ2 with and without anti-YKL-40 and secondary antibody. Left panel: MΦ1 with secondary antibody (purple) and MΦ1 with anti-YKL-40 and secondary antibody (black); middle panel: MΦ2 with secondary antibody alone (purple) and MΦ2 with anti-YKL-40 and secondary antibody (black); right panel: MΦ1 and anti-YKL-40 with secondary antibody (red) and MΦ2 and anti-YKL-40 with secondary antibody (blue). c: YKL-40 secretion in MΦ1 and MΦ2 after 24 and 48 h of stimulation with medium, LPS, TNFα and oncostatin M (OSM). Data represent mean and SEM of 4 and 3 donors (24 and 48 h, respectively). *: p < 0.001 between MΦ1 and MΦ2 for corresponding stimulus and time. $: p < 0.001 between corresponding stimulus at different time points. d: Immunofluorescence staining on sputum cytospin of a COPD patient (blue: DAPI; red: YKL-40; green: CD68)

Fig. 3

IL-12p40 secretion is dose-dependently inhibited by dexamethasone when added on day 0. IL-10 and IL-12p40 secretion with addition of dexamethasone at day 0, 3 and 7 of differentiation (0.1, 0.3 and 1nM) (a and b, left, middle and right panel, respectively). Data represent means and SEM (n = 3 donors). * p ≤ 0.05, ** p < 0.01 or *** p < 0.001: between MΦ1 to MΦ2 with corresponding stimulus. $ p ≤ 0.05 or $ p < 0.001 between MΦ1 at different concentrations of dexamethasone

Fig. 4

YKL-40 expression and protein secretion is inhibited by dexamethasone. YKL-40 mRNA levels (normalized expression) and YKL-40 protein secretion (a and b, respectively) in MΦ1 and MΦ2 with addition of dexamethasone at day 0, 3 and 7 of differentiation (0.1, 0.3 and 1nM) (left, middle and right panel, respectively). LL-37 mRNA levels (normalized expression) after 0, 3 and 7 days of differentiation with dexamethasone (0.1, 0.3 and 1nM) (c left, middle and right panel, respectively). Data represent means and SEM (n = 3 donors). * p ≤ 0.05, ** p < 0.01 or *** p < 0.001 compared to MΦ2 with corresponding stimulus. $ = p ≤ 0.05, $$ p < 0.01 or $$$ p < 0.001 compared to corresponding unstimulated cells. ##: p < 0.01 or ### p < 0.001 compared to corresponding LPS stimulated cells

Fig. 5

YKL-40 protein secretion and mRNA expression is inhibited by the demethylating agent 5AZA. IL-10 and IL-12p40 secretion after stimulation with 5-AZA (concentration 0.1, 1 and 10 μM) (a and b, respectively). YKL-40 protein secretion and mRNA expression in MΦ1 and MΦ2 after culturing with 5-AZA (concentration 0.1, 0.3 and 1nM) (c and d, respectively). Differentiated cells are stimulated with 100 ng/ml LPS for 24 h. Data represent mean and SEM (n = 3 donors). * p ≤ 0.05, ** p < 0.01 or *** p < 0.001 compared to MΦ2 with corresponding stimulus. # p ≤ 0.05, ##: p < 0.01 or ### p < 0.001 compared to corresponding LPS stimulated cells

Fig. 6

Sputum and serum YKL-40 protein levels of COPD patients before and after ICS treatment. YKL-40 levels in sputum and serum at baseline (0) and after 30 months (30 m) of inhaled corticosteroids (ICS) and placebo. For comparison of the levels between baseline and 30 months, we only included patients from whom samples were available at both time points (paired data). Each dot represent a single patient, red horizontal bars represent medians