Major Outer Membrane Protein from Legionella pneumophila Inhibits Phagocytosis but Enhances Chemotaxis of RAW 264.7 Macrophages by Regulating the FOXO1/Coronin-1 Axis

Abstract

Legionella pneumophila is an intracellular pathogen that can cause Legionnaire's disease by invading alveolar epithelial cells and macrophages. The major outer membrane protein (MOMP) plays an important role in the interaction between bacteria and host cells. However, the role of MOMP in the process of L. pneumophila invasion of macrophages and its working mechanism remain unknown. We aimed to explore the effects of MOMP on phagocytosis and chemotaxis of RAW 264.7 macrophages. The chemotactic activity, toxicity, and phagocytosis of RAW 264.7 cocultured with different concentrations of MOMP were determined by Transwell, CCK-8, and neutral red uptake assays, respectively. Target genes were detected by double-luciferase and pull down assays. qRT-PCR and Western blot were performed to analyze the expression of several important proteins involved in the immune response pathway, including coronin-1, interleukins (IL-10), forkhead transcription factor 1 (FOXO1), nucleotide-binding oligomerization domain protein (NOD) 1, NOD2, and receptor-interacting protein (RIP) 2. After coculturing with MOMP, cytological observation indicated a decrease of phagocytosis and a marked increase of chemotaxis in RAW 264.7 macrophages. The phagocytosis degree of RAW 264.7 macrophage varied with the concentration gradient of MOMP in a time-dependent manner. MOMP could increase the expression levels of MCP-1, IL-10, NOD2, and RIP2 and decrease the expression levels of FOXO1 and coronin-1 in cell culture supernatants. In addition, we found that FOXO1 could promote its transcription by binding to the promoter of coronin-1. The results of the present study suggested that MOMP could inhibit phagocytosis and facilitate chemotaxis of RAW 264.7 macrophage, which might be associated with the FOXO1/coronin-1 axis.

Figure 1

MOMP inhibits the proliferation and phagocytic activity of RAW264.7. (a) A dose-response study of MOMP indicated IC50 as 4.05 μg/ml. The curve was fitted using relative survival fraction of six working concentrations (10, 5, 2.5, 1.25, 0.625, and 0.3125 μg/ml). (b) The proliferating capacity of RAW 264.7 upon the treatment of MOMP. Statistical analysis by one-way ANOVA, ^##P < 0.01. Error bars = 95% confidence intervals. (c) The cytological phagocytosis of RAW 264.7 by neutral red uptake assay at 24 h in the four groups (control; cells treated with 0.2 μg/ml MOMP; cells treated with 0.4 μg/ml MOMP; and cells treated with 0.8 μg/ml MOMP) (original magnification: 200x, scale bar = 100 μm), P < 0.05 indicated significant differences in neutral red uptake of cells between control and treated groups. (d) Semiquantitative analysis of the phagocytosis of RAW 264.7. The significant differences were marked by the alphabetic notation (a, b, c, and d). In each same time point of different concentration treatment groups, if any two histograms with the same marking letter represented there have no significant difference between two concentration treatments, any two histograms without the same marking letter represented there have a significant difference.

Figure 2

MOMP enhances the chemotaxis of RAW264.7 (a) The chemotactic potential of RAW 264.7 cells that migrated across the membrane in the MOMP-free medium, 0.2, 0.4, and 0.8 μg/ml MOMP (crystal violet staining, original magnification: 200x, scale bar = 100 μm). (b) Chemotaxis index of RAW 264.7. (c) Secretion level of IL-10 after the treatment of different doses of MOMP for 24 h. (d) Secretion level of MCP-1 after the treatment of different doses of MOMP for 24 h (P < 0.05, error bars = 95% CIs.). The significant differences were marked by a pound sign. P values: ^#P < 0.05, ^##P < 0.01.

Figure 3

Analysis of mRNA expression of 7 proteins in RAW 264.7 treated with MOMP. The significant differences in the expression of 7 proteins in groups treated with different concentrations were marked by the alphabetic notation (a, b, c, and d). In each detected expression of 7 proteins, if any two histograms with the same marking letter represented no significant difference between two concentrations, any two histograms without the same marking letter represented a significant difference. (a–g) Relative mRNA expression analysis of NOD1, NOD2, IL-10, RIP, MCP-1, FOXO1, and coronin-1 for 24 h treated by different concentrations of MOMP. Control represented the cells cultured in a normal medium. 0.2 represented the cells cultured in the medium containing 0.2 μg/ml MOMP, 0.4 represented the cells cultured in the medium containing 0.4 μg/ml MOMP, and 0.8 represented the cells cultured in the medium containing 0.8 μg/ml MOMP (error bars = SD).

Figure 4

Expression analysis of 7 proteins at the protein level in RAW 264.7 treated by MOMP for 24 h. The significant differences in the expression of 7 proteins in groups treated with different concentrations were marked by the alphabetic notation (a, b, c, and d). In each detected expression of 7 proteins, if any two histograms with the same marking letter represented no significant difference between two concentration treatments, any two histograms without the same marking letter represented a significant difference. Relative protein expression analysis in each group was performed on the basis of the gray value of internal control β-actin. (a) Western blotting analysis of 7 proteins and β-actin. (b–h) Relative protein expression of FOXO1, MCP-1, coronin-1, IL-10, NOD1, NOD2, and RIP2 for 24 h treated by different concentrations of MOMP. Control represented the cells cultured in the normal medium; 0.2 represented the cells cultured in the medium containing 0.2 μg/ml MOMP; 0.4 represented the cells cultured in the medium containing 0.4 μg/ml MOMP; 0.8 represented the cells cultured in the medium containing 0.8 μg/ml MOMP (error bars = SD).

Figure 5

The transcription factor FOXO1 binds specifically to the promoter region of coronin-1. (a) Western blotting analysis results of DNA pull down assay. (b) Relative luciferase activity of cotransfection of different plasmids. Coronin1-pro+FOXO1 represented cotransfection of pGL3-coronin1-pro-luc+ and pcDNA3.1(+)-FOXO1; coronin1-pro represented the cells just transfected by pGL3-coronin1-pro-luc; FOXO1 represented the cells just transfected by pcDNA3.1(+)-FOXO1. The significant differences between relative luciferase activity of coronin1-pro+FOXO1 and coronin1-pro were marked by the pound sign. P values: ^##P < 0.01.