Serotonin receptor 5-HT7 modulates inflammatory-associated functions of macrophages

Abstract

The hormone and neurotransmitter serotonin regulates numerous physiological functions within the central nervous system and in the periphery upon binding to specific receptors. In the periphery, the serotonin receptor 7 (5-HT7R) is expressed on different immune cells including monocytes and macrophages. To investigate the impact of 5-HT7R-mediated signaling on macrophage properties, we used human THP-1 cells and differentiated them into pro-inflammatory M1- and anti-inflammatory M2-like macrophages. Pharmacological 5-HT7R activation with the specific agonist LP-211 especially modulates morphology of M1-like macrophages by increasing the number of rounded cells. Furthermore, 5-HT7R stimulation results in significantly reduced phagocytic and migratory ability of M1-like macrophages. Noteworthy, LP-211 treatment leads to changes in secretory properties of all macrophage types with the highest effects obtained for M0- and M2c-like macrophages. Finally, the importance of 5-HT7R for regulation of phagocytosis was confirmed in human primary CD14⁺ cells. These results indicate that 5-HT7R activation selectively impairs basic functions of macrophages and might thus be a new access point for the modulation of macrophage responses in the future treatment of inflammatory diseases.

Keywords: 5-HT7 receptor; Macrophages; Phagocytosis; Serotonin; THP-1 cells.

Fig. 1

Basal characterization of THP-1-derived macrophage subtypes. A: Differentiation scheme of THP-1-monocytes into M0-, M1-, M2a-, and M2c-like macrophages. B: Representative morphology of differentiated macrophages, arrow indicates stretched cells, * highlights enlarged cells. C: mRNA expression levels of macrophage markers. Statistical significance was evaluated using Kruskal-Wallis with Dunn’s multiple comparison test (N = 5 independent differentiations). D: Quantitative analysis of surface marker expression of CD11b, HLA-DR, CD86, and MERTK for each macrophage subtype using flow cytometry. Fluorescence intensity shifts are displayed with normalized counts; u: unstained control. Fold changes of median fluorescence intensities (MFI) relative to M0-like macrophages were analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test (CD11b, HLA-DR, CD86) and Kruskal-Wallis with Dunn’s multiple comparison test (MERTK) (N ≥ 3 independent differentiations)

Fig. 2

THP-1-derived macrophages express the 5-HT7R and downstream signaling molecules. A: mRNA levels of 5-HT7R and Gαs, Gα12, and Cdc42. Statistical difference was evaluated using Kruskal-Wallis with Dunn’s multiple comparison test (N ≥ 5). B: Protein expression levels of 5-HT7R and its downstream effectors shown by western blot. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons (5-HT7R, Gα12) and Kruskal-Wallis with Dunn’s multiple comparison (Gαs, Cd42) test (N = 5). C: Representative images visualizing 5-HT7R localization. Antibody specificity was confirmed with a corresponding blocking peptide (right panel; scale bars 20 μm)

Fig. 3

5-HT7R activation influences macrophage morphology. A: Characterization of macrophages morphology using plasma membrane staining in living cells. Representative images of cell shape on day 5 of differentiation are shown. Scale bars 50 μm in upper panel and 20 μm in magnification. B: Relative quantification of cell shape by counting round, stretched and enlarged phenotypes after THP-1 differentiation. C: Representative images of THP-1-derived macrophage morphology following LP-211 treatment (10 µm; 48 h). Scale bars 50 μm in upper panel and 20 μm in magnification (N = 3). D: Quantification of morphology profiles after treatment with LP-211 (10 µm; 48 h). E: Quantification of protrusion numbers in differentiated macrophages without and after LP-211 (10 µm; 48 h) treatment. Statistical analysis was performed on daily mean values (N = 3) using two-way ANOVA with Tukey’s multiple comparisons test. F: Analysis of mean length of protrusions under basal conditions and after 5-HT7R stimulation with LP-211 (10 µm; 48 h). Statistical analysis was performed on daily mean values (N = 3) using two-way ANOVA with Tukey’s multiple comparisons test

Fig. 4

Motility of macrophages is influenced by 5-HT7R activity. A: Representative images of scratch area at beginning (0 h) and 24 h after scratch initiation for all macrophage subtypes. Scale bars 200 μm. B: Recovered area of macrophage subtypes relative to scratch area at 0 h. Quantification of migratory ability after 24 h. Data are presented as mean with standard deviation. Adjusted p values: † M0 vs. M1 p = 0.0004, ⁺ M0 vs. M2a p = 0.0084, ^# M1 vs. M2c p = 0.002, and M2a vs. M2c p = 0.0398 using one-way ANOVA with Tukey’s multiple comparisons test (N = 4). C: Images of scratch area at 0 h and 24 h after LP-211 treatment (10 µm). Scale bars 200 μm. D: Quantification of macrophage migration ability after 24 h after LP-211 (10 µm) or control treatment. Statistical significance was evaluated using unpaired Mann-Whitney test between control and LP-211 conditions of each macrophage subtype (N = 5)

Fig. 5

Phagocytosis rate of M1-like macrophages is reduced upon 5-HT7R activation. A: Representative images of DAPI stained nuclei (upper row) and Texas red-coupled Zymosan A particles (lower row) in control-treated macrophages. Scale bars 200 μm. B: Fraction of phagocytic macrophages showing engulfment of Texas red-coupled Zymosan A particles. Statistical differences are evaluated using ordinary one-way ANOVA with Tukey’s multiple comparison test (N = 8; each point displays the mean of 3 replicate wells). C: Representative images of macrophages treated with LP-211 (10 µm) showing DAPI and Texas red-coupled Zymosan A particle fluorescence after phagocytosis assay. Scale bars 200 μm. D: Quantification of phagocytosis rate of macrophage subtypes with control or LP-211 treatment (10 µm; 48 h). Statistical significance was determined using unpaired two-tailed t-test (N = 8; each point displays the mean of 3 replicate wells)

Fig. 6

Modulation of secretion ability of macrophages upon 5-HT7R stimulation. A: Relative secretion profile in the supernatant of THP-1-derived macrophages by normalization to protein concentration (N=3). Arrows indicate concentrations above the highest value of the standard. B: Relative fold changes of secreted proteins after LP-211 (10 µm) treatment starting day 3 after seeding compared to control. C: Principal component analysis of three independent differentiations (one dot displays one differentiation) using the first two principal components (N=3). D: Principal component analysis to visualize clustering under basal conditions and after LP-211 treatment for each macrophage subtype. Each principal component was assessed in a separated analysis using Kaiser’s rule (N=3)

Fig. 7

Impact of 5-HT7R signaling on primary human CD14⁺ macrophages. A: mRNA expression levels of differentiation markers in CD14⁺ cells. Statistical significance was evaluated using ordinary one-way ANOVA with Tukey’s multiple comparisons test (N = 6 independent differentiations/donors). B: mRNA expression level of 5-HT7R at day 7 of differentiation. Statistical difference was evaluated using ordinary one-way ANOVA with Tukey’s multiple comparisons test (N = 6). C: Representative images of DAPI stained nuclei (upper row) and Texas red-coupled Zymosan A particles (lower row) in CD14⁺-derived macrophages after 24 h phagocytosis assay under basal conditions. Scale bars 200 μm. D: Quantification of phagocytic cells showing engulfment of Texas red-coupled Zymosan A particles under basal conditions. Statistical differences were evaluated using ordinary one-way ANOVA with Tukey’s multiple comparison test (N = 4; each point displays the mean of ≥ 4 replicate wells). E: Representative images of macrophages treated with LP-211 (10 µM) showing DAPI and Texas red-coupled Zymosan A particle fluorescence after phagocytosis assay. Scale bars 200 μm. F: Quantification of phagocytosis rate of macrophage subtypes with control (DMSO) or LP-211 treatment (10 µM; 48 h). Statistical significance was determined using paired two-tailed t-test (N = 4; each point displays the mean of ≥ 4 replicate wells)