Fig 1.
DBV-infected macrophages exhibit pyroptosis-specific cell morphology and cell death.
PMA-activated THP-1 cells were incubated on coverslips in six-well plates with different concentrations of DBV (MOI = 0.1, 1), LPS and nigericin, or culture medium. After 72 h of incubation, unbound cells were washed out, and images were obtained by (A) scanning electron microscopy and (B) inverted microscopy analysis at a magnification of ×200. The red arrow indicates classical morphological features of pyroptosis. Representative images of three replicates per macrophage group were shown. PMA-activated THP-1 cells were treated with different doses of DBV (MOI = 0.01, 0.1, 1) for 72 h incubation, or infected with DBV at MOI of 1 for 24 h, 48 h, 72 h and 96 h incubation, or treated with LPS and nigericin, or were mock infected. (C and D) After incubation at indicated time points, cell viability was detected by the CCK-8 kit. (E and F) Supernatants from different coculture conditions were collected, and cell death was determined using an LDH release assay. Data were mean values ± SD derived from the samples collected in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with mock; #P<0.05, ##P<0.01, ###P<0.001 compared between different treatments, by one-way ANOVA test.
Fig 2.
DBV infection induces pyroptosis characterized by caspase-1 and GSDMD activation and IL-1β secretion.
(A and B) Relative expression levels of caspase-1 mRNA in macrophages were quantified by RT-qPCR. (C and D) Relative expression levels of GSDMD mRNA in macrophages were quantified by RT-qPCR. (E and F) Relative expression levels of IL-1β mRNA in macrophages were quantified by qPCR. A to F: Data were mean values ± SD derived from the samples collected in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with mock; #P<0.05, ##P<0.01, ###P<0.001 compared between different treatment conditions, by one-way ANOVA test. (G) Macrophages were infected with DBV at different MOIs (0.01, 0.1, 1). At indicated time points, supernatants from different coculture conditions were collected, and IL-1β levels were measured by ELISA kit. Data were mean values ± SD derived from the samples collected in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with MOI = 0.01, by one-way ANOVA test. (H) Examination of the proteolytic cleavage of caspase-1, GSDMD and IL-1β in human macrophages with DBV or mock infection using western blot analysis (72 h.p.i). β-actin was probed as a loading control.
Fig 3.
Platelet addition altered the transcriptome of DVB-infected macrophages.
Macrophages were infected with DBV at MOI of 1 for 72h as a “no platelet” group (n = 4), or infected with DBV at MOI of 1 with platelets for 72h as a “platelet added” group (n = 4). (A) Volcano plot analysis of mRNAs differentially expressed in response to the “platelet added” group vs “no platelet” group. Red dots indicate up-regulated genes and blue dots indicate down-regulated genes. (B) Heatmap of significantly differentially expressed macrophage transcripts from the “no platelet” group and “platelet added” group. Red represents enhanced relative expression, and blue represents reduced relative expression. (C) GO enrichment analysis of differentially expressed mRNAs for no-platelets and platelet-added macrophages. (D and E) IL-1β mRNA in macrophages was quantified by RT-qPCR. Data were mean values ± SD derived from the samples collected in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with mock; #P<0.05, ##P<0.01, ###P<0.001 compared between different treatment conditions, by one-way ANOVA test.
Fig 4.
DBV-mediated macrophage pyroptosis and inflammatory responses are attenuated by platelets.
Macrophages were mock infected, or treated with LPS and nigericin, or infected with DBV at MOIs of 0.01, 0.1, 1, or co-incubated with platelets and DBV at MOIs of 0.01, 0.1, 1. (A) After incubation at required time points, cell viability was measured by the CCK-8 kit. (B, C) caspase-1 mRNA in macrophages from different coculture conditions were quantified by RT-qPCR. (D, E) GSDMD mRNA in macrophages from different coculture conditions was quantified by RT-qPCR. A to E: Data were mean values ± SD derived from the samples collected in triplicate. *P<0.05, **P<0.01, ***P<0.001 compared with mock; #P<0.05, ##P<0.01, ###P<0.001 compared between different treatment conditions, by one-way ANOVA test. (F-I) IL-1β levels in supernatants were determined by ELISA kit. *P<0.05, **P<0.01, ***P<0.001 compared with MOI = 0.01, by one-way ANOVA test. (J) NLRP3, caspase-1, GSDMD, pro IL-1β as well as their cleaved proteins in the cell lysates were determined by western blot. β-actin was probed as a loading control.
Fig 5.
Model depicting DBV-mediated pyroptosis and inflammatory responses in macrophages.
In SFTS, DBV virions are phagocytosed by human macrophages in the circulation. DBV takes advantage of human macrophages for its own replication but triggers the activation of inflammasome NLRP3. NLRP3 inflammasome activation then induces the auto-cleavage of caspase-1. Once assembled, caspase-1 mediates the proteolytic process of GSDMD and IL-1β, triggering pyroptosis to facilitate host defense (right). On the contrary, in the presence of platelets, DBV-infected macrophages show suppressed activation of caspase-1 and GSDMD and reduced inflammatory response related to IL-1β in the same process, protecting human macrophages from pyroptosis (left).