MC-derived serotonin promotes DENV-induced thrombocytopenia. (A) Serotonin was detected by immunostaining of control BMMCs, whereas activation of BMMCs and release of serotonin was observed after incubation with DENV2 at a multiplicity of infection of 1 for 2 hours. Scale bar, 20 μm. (B) BMMCs release serotonin into the cell culture supernatant after DENV exposure as in panel A, measured by enzyme-linked immunosorbent assay. (C) WT and Sash mice were infected with DENV2 (1 × 10⁶ PFU via intraperitoneal injection). At 24 hours postinfection, plasma was isolated and serotonin levels were measured by enzyme-linked immunosorbent assay. (D-F) Mice selectively deficient in MC serotonin were generated through reconstitution of Sash mice with BMMCs deficient in TPH-1 (Sash-R^TPH1-KO). For controls, mice were reconstituted with BMMCs from WT mice (Sash-R^WT). A schematic of the experimental strategy is provided in supplemental Figure 4A. (D) Platelet counts decreased significantly in Sash-R^WT infected with DENV2, but not in infected Sash-R^TPH1-KO mice at day 3 postinfection (n = 4-7 per group). (E) Increased numbers of activated platelets (CD41⁺CD62P⁺) were detected in the circulation of DENV-infected Sash-R^WT mice, but not in infected Sash-R^TPH1-KO compared with uninfected controls. Flow cytometry gating strategy is presented in supplemental Figure 2A (n = 7-9 per group). (F) Representative eosin and methylene blue staining of peripheral blood smears day 3 postinfection. Some individual platelets are indicated by arrows. Platelet aggregates are surrounded by a dashed line. Scale bar, 25 μm. (G) Sash mice were infected with DENV2 and treated with 150 mg/kg serotonin or vehicle daily. Blood was taken for analysis 3 days postinfection. In infected animals, treatment with serotonin significantly reduced circulating platelet counts compared with vehicle treatment zanimals. (H) Serotonin treatment resulted in increased platelet activation in infected mice compared with vehicle-treated controls (n = 5-8 per group). Means are presented with error bars representing the standard error of the mean. P values were determined by 1-way ANOVA for panels C-D or by Student’s unpaired t test for panels B, F-G. ns, not significant; *P < .05; **P < .01; ***P < .001; ****P < .0001.