SAA drives proinflammatory heterotypic macrophage differentiation in the lung via CSF-1R-dependent signaling

Abstract

Serum amyloid A (SAA) is expressed locally in chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD), where macrophages that do not accord with the classic M1/M2 paradigm also accumulate. In this study, the role of SAA in regulating macrophage differentiation was investigated in vitro using human blood monocytes from healthy subjects and patients with COPD and in vivo using an airway SAA challenge model in BALB/c mice. Differentiation of human monocytes with SAA stimulated the proinflammatory monokines IL-6 and IL-1β concurrently with the M2 markers CD163 and IL-10. Furthermore, SAA-differentiated macrophages stimulated with lipopolysaccharide (LPS) expressed markedly higher levels of IL-6 and IL-1β. The ALX/FPR2 antagonist WRW4 reduced IL-6 and IL-1β expression but did not significantly inhibit phagocytic and efferocytic activity. In vivo, SAA administration induced the development of a CD11c(high)CD11b(high) macrophage population that generated higher levels of IL-6, IL-1β, and G-CSF following ex vivo LPS challenge. Blocking CSF-1R signaling effectively reduced the number of CD11c(high)CD11b(high) macrophages by 71% and also markedly inhibited neutrophilic inflammation by 80%. In conclusion, our findings suggest that SAA can promote a distinct CD11c(high)CD11b(high) macrophage phenotype, and targeting this population may provide a novel approach to treating chronic inflammatory conditions associated with persistent SAA expression.

Keywords: ALX/FPR2 signaling; COPD; lung inflammation; macrophage biology.

Figure 1.

SAA initiates expression of inflammatory genes and CD163 in primary blood monocytes. A–C) Representative images of monocytes at d 0 (A) and MDMs following differentiation over 7 d (D7) in the (B) absence (−SAA; B) or presence of SAA (+SAA; C). D–F) At d 7, gene expression of IL-6 (D), IL-1β (E), and CD163 (F) was determined in the MDM populations. Data represent n = 4 individual blood samples. *P < 0.05.

Figure 2.

IL-10 regulates CD163, but not IL-6, in COPD MDMs generated in the presence of SAA. A, B) Gene expression of IL-6 (A) and CD163 (B) was compared between MDMs generated from healthy controls and subjects with COPD. C, D) Spearman correlation between IL-10 and IL-6 (C) and IL-10 and CD163 (D) in SAA-induced MDMs from patients with COPD. E, F) No relationship was observed between systemic serum SAA levels and expression of IL-6 (E) or CD163 (F). Data represent n = 6 for control and n = 7 for COPD individual blood samples. *P < 0.05 vs. vehicle-treated MDM expression; ^#P < 0.05; Spearman test.

Figure 3.

SAA induces increased phagocytosis and efferocytosis in primary human MDMs. A, B) Representative histograms show phagocytosis of E. coli (A) and apoptotic lymphocytes (B) by primary MDMs in the absence (dotted line) or presence of SAA (solid line). C, D) Quantification of phagocytic (C) and efferocytic (D) activity of MDMs from individual donors in the absence (vehicle) or presence of SAA. Data from n = 5–6 samples. *P < 0.05.

Figure 4.

Targeting ALX/FPR2 inhibits inflammatory gene expression without compromising macrophage function. Expression of IL-6 and IL-1β was determined in SAA-induced MDMs in the absence or presence of WRW4. WRW4 reduced (A) IL-6 and (B) IL-1β by 37% and 38%, respectively. On analysis of (C) phagocytosis and (D) efferocytosis in primary human MDMs generated with SAA, no significant inhibition of function was observed. Data represent n = 4 individual blood samples, *P < 0.05.

Figure 5.

SAA-generated MDMs express higher IL-1β and IL-6 in response to LPS. SAA-induced MDMs were stimulated with or without LPS (1 μg/ml) for 2 h, and expression of IL-6 (A) and IL-1β (B) was determined by qPCR. Gene expression is expressed relative to the housekeeping gene 18S and presented by fold change above vehicle treatment (VEH). Data represent n = 4 individual blood samples.

Figure 6.

Acute rSAA treatment into murine lung results in the emergence of CD11c^highCD11b^high macrophage population. CD11c and CD11b expression was used to determine macrophage subsets. A, B) Representative dot plots of macrophage subpopulations in BAL fluid 48 h after vehicle (A) or SAA treatment (B). C, D) Number of CD11c^highCD11b^low cells per milligram of lung tissue (C) and BAL (D). E, F) Number of CD11c^highCD11b^high cells per milligram of lung tissue (E) and BAL (F). Pooled data from 2 experiments n = 4–8 samples. *P ≤ 0.05.

Figure 7.

Chronic administration of rSAA results in the sustained presence of the CD11c^highCD11b^high population that expresses high levels of IL-6, IL-1β, and G-CSF. A) Percentage of CD11c^highCD11b^high macrophages in the BAL compartment. B–D) Macrophage populations were isolated from lung tissue using a cell sorting strategy and stimulated ex vivo with saline (control) or LPS. Expression of IL-6 (B), IL-1β (C), and G-CSF (D) were determined by quantitative PCR and expressed as a fold increase above resident CD11c^highCD11b^low macrophages. Pooled data from 2 experiments; n = 4–8 samples. *P ≤ 0.05.

Figure 8.

The emergence of SAA-induced CD11C^hiCD11b^hi macrophage subpopulation is dependent on CSF-1R signaling. BALB/c mice were intranasally pretreated with anti-CSF-1R or control followed by rSAA. BAL cells were analyzed by flow cytometry 24 h later for macrophage subset populations. Neutrophils were identified as CD11c⁻Ly6G⁺ cells. Graphs show total number of CD11c^highCD11b^low macrophages (A), CD11c^highCD11b^high macrophages (B), and neutrophils (C); n = 4–6 individual samples/group. *P ≤ 0.05.