Neuroendocrine signaling via the serotonin transporter regulates clearance of apoptotic cells

Abstract

Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.

Keywords: Autoimmune Diseases; Efferocytosis; Inflammation; Lung; Phagocytosis; Rho Kinase; RhoA; Serotonin Receptors.

FIGURE 1.

5-HT impairs ingestion of apoptotic cells and carboxylated beads by murine peritoneal macrophages. A and B, photomicrographs (40x original magnification, bar = 5 μm) show binding (arrows) and ingestion (arrowheads) of apoptotic thymocytes by murine peritoneal macrophages. C and D, murine resident (C) and TG-elicited (D) peritoneal macrophages were treated with 5-HT at 1, 10, 100, and 1,000 nm for 24 h then apoptotic thymocytes were co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO₂ at 37 °C. Uningested apoptotic thymocytes were then washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 3 per group. *, one-way ANOVA, p < 0.05 versus media control (Dunnett's Test); **, p < 0.01 versus media control (Dunnett's test). E and F, murine resident (E) and TG-elicited (F) peritoneal macrophages were treated with 5-HT at 1, 10, 100, and 1,000 nm for 24 h, then carboxylated beads were co-cultured with macrophages at a 2:1 ratio for 20 min in 10% CO₂ at 37 °C. Uningested beads were washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 3 per group. *, one-way ANOVA, p < 0.05, p < 0.05 versus media control (Dunnett's test).

FIGURE 2.

5-HT impairs phagocytosis of latex beads by murine peritoneal macrophages, but has no effect on uptake of IgG-opsonized erythrocytes through the Fcγ receptor. A and B, mouse resident (A) and TG-elicited (B) peritoneal macrophages were treated with 5-HT at 1, 10, 100, and 1,000 nm for 24 h, then latex beads were co-cultured at a 1:1 ratio for 10 min in 10% CO₂ at 37 °C. Uningested beads were washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 3 per group, *, one-way ANOVA, p < 0.05 versus media control (Dunnett's Test). **, p < 0.01 versus media control (Dunnett's test). C and D, mouse resident (C) and TG-elicited (D) peritoneal macrophages were treated with 5-HT at 1, 10, 100, and 1,000 nm for 24 h then co-cultured with IgG-opsonized erythrocytes (RBCs) at a 10:1 ratio in 10% CO₂ at 37 °C. Uningested RBCs were then washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 3 per group.

FIGURE 3.

5-HT impairs efferocytosis through activation of the RhoA/Rho kinase pathway. A, murine TG-elicited macrophages were treated with 5-HT at 1,000 nm for 24 h. The RhoA inhibitor, C3 transferase (1 μg/ml), was added either alone or in the presence of 5-HT for 3 h prior to the assay. Apoptotic thymocytes were co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO₂ at 37 °C. Uningested apoptotic thymocytes were washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 3 per group. *, one-way ANOVA, p < 0.05 versus media control (Dunnett's test). B, murine TG-elicited macrophages were treated with 5-HT at 100 or 1,000 nm for 24 h. The ROCK inhibitor, Y-27632 (10 μm), was added either alone or in the presence of 5-HT for 3 h prior to the assay. Apoptotic thymocytes were co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO₂ at 37 °C. Uningested apoptotic thymocytes were washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 3 per group. **, one-way ANOVA, p < 0.05, p < 0.01 versus media control (Dunnett's test). C, murine TG-elicited peritoneal macrophages were stimulated with 5-HT (1 μm) for the indicated times. Phosphorylation of myosin phosphatase 1 (Mypt1) at Thr-696 increased upon 5-HT stimulation. Representative immunoblots are shown in the upper panel. In the lower panel, densitometry was used to determine the ratio of phospho-Mypt1 to total Mypt1. Data represent the mean ± S.E. as a percent of media control for n = 3 per group. Media control = 0.2545 ± 0.1182. *, F-test, p < 0.05 for significance of percent difference from media control (Bonferroni t statistic). D, murine TG-elicited peritoneal macrophage were stimulated with 5-HT (1 μm) for the indicated times in the presence and absence of the Rho kinase inhibitor, Y-27632 (10 μm), and examined for the ratio of phospho-Mypt-1 to total Mypt1 by Western blot. Data represent the mean ± S.E. as percent of media control for n = 5 per group. Media control = 0.1807 ± 0.0286. *, F-test, p < 0.05 for significance of percent control from media control (Bonferroni t statistic).

FIGURE 4.

5-HT receptor 2a, 2b, and the 5-HT transporter are expressed in mouse peritoneal macrophages. A, real-time PCR was performed to measure the expression by mRNA. 5-HT receptor 2a (5HTR2a), 5-HT receptor 2b (5HTR2b), and the 5-HTT were expressed in TG-elicited peritoneal macrophages, but 5-HT receptor 2c (5HTR2c) was not detected. Data represent the mean ± S.E. for n = 3 per group. B–D, Western blotting is shown for two representative peritoneal macrophage lysates (PM1 and PM2) evaluating expression of 5-HT receptors and transporter. B, HTR2a (n = 4); C, HTR2b (n = 10); and D, 5-HTT (n = 8) were all detected in mouse peritoneal macrophages.

FIGURE 5.

5-HT utilizes the 5-HT transporter, but not 5-HT receptors 2a or 2b, to regulate efferocytosis by murine peritoneal macrophages. A and B, TG-elicited macrophages were treated with 5-HT at 1,000 nm in the presence and absence of: A, the 5-HT receptor 2b inhibitor, R-96544; B, the 5-HT receptor 2b inhibitor, RS-127445; or C, the 5-HT transporter inhibitor, fluoxetine, at the indicated concentrations for 24 h. Apoptotic thymocytes were co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO₂ at 37 °C. Uningested apoptotic thymocytes were washed off and a PI was determined by blinded visual inspection. 5-HT does not inhibit efferocytosis via 5HTR2a- or 5HTR2b-dependent pathways. Data represent the mean ± S.E. for n = 4–5 replicates per group, *, one-way ANOVA, p < 0.05 versus DMSO control (Dunnett's test); **, p < 0.01 versus DMSO control (Dunnett's test). C, TG-elicited macrophages were treated with 5-HT at 1.0 μm for 24 h in the presence and absence of the 5-HT transporter inhibitor, fluoxetine, at 0.1 and 1.0 μm. Apoptotic thymocytes were then co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO₂ at 37 °C. Uningested apoptotic thymocytes were washed off and a PI was determined by blinded visual inspection. Data represent the mean ± S.E. for n = 5 per group, *, one-way ANOVA, p < 0.05 versus media control (Dunnett's test).

FIGURE 6.

Effect of 5-HTT deficiency on 5-HT uptake, efferocytosis, Rho kinase activity, and peritoneal inflammation. A, function of the 5-HTT in TG-elicited peritoneal macrophages was investigated using a neuroendocrine uptake assay, where a tracer becomes fluorescent upon internalization. The tracer can be internalized by the 5-HT, dopamine, or the norepinephrine transporters, so specificity depends on genetic deletion of a given transporter. TG-elicited peritoneal macrophages were isolated from 5-HTT KO (gray circles) and wild-type control mice (black circles), and uptake of the fluorescent tracer was assessed and expressed as relative fluorescent units (RFU). Lines indicate paired experiments. p = 0.016, wild-type versus 5-HTT KO mice (paired t test). B, TG-elicited peritoneal macrophages from wild-type mice (black columns) and 5-HTT KO mice (gray columns) were treated with media alone or media containing 5-HT (1 μm) for 24 h. Apoptotic thymocytes were co-cultured with macrophages at a 10:1 ratio for 1 h in 10% CO₂ at 37 °C. Uningested apoptotic thymocytes were washed off and a PI was determined by blinded visual inspection. 5-HT decreased efferocytosis in wild-type macrophages. Data represent the mean ± S.E. for n = 8 per group. Two-way ANOVA, p < 0.001 for interaction, and p < 0.01 for treatment effect (media versus 5-HT treatment). *, Tukey-Kramer post hoc analysis, p < 0.001 for wild-type 5-HT treatment versus wild-type media control; p < 0.01 for wild-type 5-HT treatment versus 5-HTT KO 5-HT treatment; p < 0.05 for wild-type 5-HT treatment versus 5-HTT KO media control. C, murine TG-elicited peritoneal macrophages from wild-type (black columns) and 5-HTT KO mice (gray columns) were stimulated with 5-HT (1 μm) for the indicated times. Densitometry was used to determine the ratio of phosphomyosin phosphatase 1 (pMypt1) to total Mypt1 by arbitrary units. 5-HT increased phosphorylation of Mypt1 at Thr-696 in wild-type but not in 5-HTT KO macrophages. Data represent the mean ± S.E. for n ≤ 13 per group. Two-way ANOVA, p = 0.03 for interaction, and p < 0.01 for genotype effect (wild-type versus 5-HTT KO). *, Tukey-Kramer post hoc analysis p < 0.03 for wild-type media control versus wild-type 5-HT treatment for 5 min; **, p < 0.01 for wild-type media control versus wild-type 5-HT treatment for 20 min; †, p < 0.03 for wild-type 5-HT treatment for 20 min versus 5-HTT KO 5-HT treatment for 20 min. D, 5-HTT KO (gray circles) and wild-type (black circles) mice were injected with TG. Four days later peritoneal lavage was performed and total cell counts and differentials were taken. Neutrophils were decreased in 5-HTT KO mice compared with wild-type mice. Data represent the mean ± S.E. for n = 8–10 mice per group. p = 0.036, wild-type versus 5-HTT KO mice (Mann-Whitney test).

FIGURE 7.

5-HT inhibits efferocytosis by human alveolar macrophages via a mechanism that involves the 5-HT transporter and Rho kinase. A, Western blots were performed on lysates from human alveolar macrophages from n = 5 normal subjects and probed for the presence of the 5-HT transporter. B, human alveolar macrophages were treated with 5-HT at 1.0 μm for 24 h in the presence and absence of the 5-HT transporter inhibitor, fluoxetine, at 0.1, 1.0, and 10 μm. Apoptotic human Jurkat T-cells were then co-cultured with macrophages at a 10:1 ratio for 3 h in 10% CO₂ at 37 °C. Uningested apoptotic cells were washed off and PI was determined by visual inspection. Data represent the mean ± S.E. for n = 3 per group, *, one-way ANOVA, p < 0.05 versus media control (Dunnett's test). C, human alveolar macrophages were treated with 5-HT at 1.0 μm for 24 h in the presence and absence of the ROCK inhibitor, Y-27632, at 10 μm. Apoptotic human Jurkat T-cells were then co-cultured with macrophages at a 10:1 ratio for 3 h in 10% CO₂ at 37 °C. Uningested apoptotic cells were washed off and a PI was determined by visual inspection. Data represent the mean ± S.E. for n = 4 per group, *, one-way ANOVA, p < 0.05 versus media control (Dunnett's test).

FIGURE 8.

Proposed model for the ability of 5-HT to suppress efferocytosis. 5-HT impairs efferocytosis by activating RhoA and ROCK. 5-HT appears to exert its effect on efferocytosis through the 5-HT transporter and not through 5-HT receptor 2a or 5-HT receptor 2b (red X).