Serum amyloid A opposes lipoxin A₄ to mediate glucocorticoid refractory lung inflammation in chronic obstructive pulmonary disease

Abstract

Chronic obstructive pulmonary disease (COPD) will soon be the third most common cause of death globally. Despite smoking cessation, neutrophilic mucosal inflammation persistently damages the airways and fails to protect from recurrent infections. This maladaptive and excess inflammation is also refractory to glucocorticosteroids (GC). Here, we identify serum amyloid A (SAA) as a candidate mediator of GC refractory inflammation in COPD. Extrahepatic SAA was detected locally in COPD bronchoalveolar lavage fluid, which correlated with IL-8 and neutrophil elastase, consistent with neutrophil recruitment and activation. Immunohistochemistry detected SAA was in close proximity to airway epithelium, and in vitro SAA triggered release of IL-8 and other proinflammatory mediators by airway epithelial cells in an ALX/FPR2 (formyl peptide receptor 2) receptor-dependent manner. Lipoxin A(4) (LXA(4)) can also interact with ALX/FPR2 receptors and lead to allosteric inhibition of SAA-initiated epithelial responses (pA(2) 13 nM). During acute exacerbation, peripheral blood SAA levels increased dramatically and were disproportionately increased relative to LXA(4). Human lung macrophages (CD68(+)) colocalized with SAA and GCs markedly increased SAA in vitro (THP-1, pEC(50) 43 nM). To determine its direct actions, SAA was administered into murine lung, leading to induction of CXC chemokine ligand 1/2 and a neutrophilic response that was inhibited by 15-epi-LXA(4) but not dexamethasone. Taken together, these findings identify SAA as a therapeutic target for inhibition and implicate SAA as a mediator of GC-resistant lung inflammation that can overwhelm organ protective signaling by lipoxins at ALX/FPR2 receptors.

Fig. 1.

Relationship between lung SAA, IL-8 and neutrophil activity. Correlation between secreted (A) SAA and NE activity; (B) IL-8 and NE, and SAA and IL-8 were determined in BALF from COPD subject (n = 41). (^δP < 0.05, Pearson correlation).

Fig. 2.

ALX/FPR2 is expressed in COPD lungs and is a functional receptor for SAA in human airway epithelial cells. (A) Representative Isotype (ISO), ALX/FPR2 (ALX) and SAA staining on serial Control (Upper) and COPD sections (Insets: 100× magnification). ALX/FPR2 inset demonstrates prominent apical and basolateral (BL) epithelial ALX/FPR2 expression in COPD. MCP-1 (B) and GM-CSF (C) levels in cell-free supernatants were measured in null- A549 cells (open bar) and rhALX- A549 cells (black bar) treated with Vehicle (VEH) or recombinant SAA (1 mg/mL) for 24 h. rhALX- A549 cells were preincubated with LXA₄ (10⁻⁸ M or 10⁻⁷ M) (D) or 15-epi-LXA₄ (10⁻⁷ M) (E) for 30 min followed by recombinant SAA (10⁻¹⁰ M to 10⁻⁷ M, 24 h). Release of IL-8 was measured in cell-free supernatants by ELISA (see SI Methods). The data are expressed as a percentage of the maximal SAA-stimulated IL-8 release and the mean ± SEM for n = 5–7 measured in duplicate, ^#P < 0.05.

Fig. 3.

Relationship between SAA and LXA₄ during AECOPD. (A) LXA₄ and (B) SAA concentration (nM) in matching blood samples from COPD subjects during (i) the acute phase of an exacerbation (AECOPD) and at (ii) clinical recovery (Resolution) (n = 10, *P < 0.05 by Wilcoxon-matched paired t test). (C) The relative ratio of SAA/LXA₄ was determined for the matching AECOPD and recovery samples. (D) The SAA/LXA₄ ratio was also determined for a control cohort with normal lung function (^#P < 0.05 by ANOVA vs. Control). The association of LXA₄ with (E) SAA and (F) LTB₄ was compared during the AECOPD phase, demonstrating a significant correlation with LTB₄ only (^δP = 0.002, r = 0.87 Pearson correlation).

Fig. 4.

Local SAA delivery promotes lung inflammation and CXCL1/2 that is suppressed by 15-epi-LXA₄. Mice were challenged with SAA (2 μg) via intranasal administration and at the indicated timepoints (h) were killed. (A) Total BAL cells and differential analysis was used to determine (B) neutrophil BAL numbers. (C) Secreted CXCL1 concentration in BALF was determined by ELISA. (D and E) CXCL1 and CXCL2 mRNA in response to SAA and the effect of concurrent 15-epi-LXA₄ (4 μg) on CXCL1/2 expression (F) and neutrophil recruitment at 6 h (G) and 24 h (H) were determined by TaqMan QPCR. (I) In a separate experiment, mice were pretreated with DEX (0.5 mg/kg i.p.) for 2 h before intranasal SAA challenge (1 μg, 24 h). (J) The effect of DEX on SAA-mediated CXCL1/2 expression in lung tissue was determined by qPCR. Data are presented at a percentage of the SAA+VEH-treated group. Results are expressed as mean ± SEM (n = 4–8) where ^#P < 0.05 by ANOVA and ^δP < 0.05 by unpaired t test.

Fig. 5.

SAA is elevated in COPD airways and colocalizes with BAL and tissue macrophages. (A) A representative specimen of Intense SAA staining in COPD. (B) Serial sections were immunostained for CD68⁺ macrophages (50× magnification). (C) SAA expression in bronchoalveolar COPD macrophages was detected by immunocytochemistry. (D) SAA expression was measured by qPCR in THP-1 cells treated with LPS (1 μg/mL) or DEX (10⁻⁶ M) for 24 h. (E) The combination of DEX and LPS was next assessed and normalized to LPS alone, which was designated as 100%. Note that because of the large synergistic effect of DEX and LPS (35,000% at 10⁻⁶ M DEX), the y axis was log-transformed. The DEX concentration dose–response curve for LPS treated THP-1 cells generated an EC₅₀ of 43 nM. (F) IL-8 levels were also measured at the DEX dose (10⁻⁶ M), causing maximal induction of SAA, demonstrating effective suppression of IL-8. The data are expressed as a percentage of the LPS response and the mean ± SEM for n = 4–5, ^#P < 0.05.