Fig 1.
PTX3 expression is elevated in CHIKF and RRVD patients.
Expression profile of PTX3 in PBMCs of (A) CHIKF patients (n = 20) or healthy controls (n = 9) were analyzed by qRT-PCR. Data were normalized to GAPDH and shown as fold expression relative to healthy controls. The CHIKF patient cohort was separated into (B) viral load groups: high viral load (HVL; n = 10) and low viral load (LVL; n = 10), and (C), disease severity group: severe (n = 10) vs mild (n = 10). (D) Serum from RRVD patients (n = 21) or healthy controls (n = 10) were analyzed by ELISA for PTX3 levels. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, Mann-Whitney U test.
Fig 2.
PTX3 expression is up-regulated following RRV infection in murine model.
(A) 21-day-old C57BL/6 WT mice (n = 4–5 per group) were subcutaneously injected with 104 PFU of RRV or PBS (mock). Mice were sacrificed at 2, 5, 10 and 15 dpi. Serum, quadriceps and ankle joints were harvested. PTX3 expression in serum of RRV- or mock-infected mice was determined by ELISA. (B) Transcriptional profile of PTX3 in quadriceps and ankle joint harvested from RRV- or mock-infected mice at various time points were determined by qRT-PCR. Data were normalized to HPRT and shown as fold expression relative to mock-infected. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA, Bonferroni post-test. Data are presented as mean ± SEM and are representative of 2 independent experiments. (C) Histology of RRV-induced inflammation in quadriceps of WT mice was analyzed by IHC staining with anti-PTX3 antibody at 10 dpi. Arrows indicate abundance of inflammatory infiltrates. Images were taken at 20× magnification. Scale bar, 40 μm. (D) 21-day-old C57BL/6 WT (n = 2–3 per group) mice were infected subcutaneously with 104 PFU RRV. Spleens were harvested at 2 dpi and were characterized and quantified by flow cytometry using the markers as described in Materials and Methods to determine mean fluorescence intensity (MFI) of PTX3 expression in total leukocytes, inflammatory monocytes, neutrophils, NK cells, T cells and B cells. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA, Bonferroni post-test.
Fig 3.
PTX3 modulates RRV replication and disease onset in mice.
(A) 21-day-old C57BL/6 WT and PTX3-/- mice were infected subcutaneously with 104 PFU RRV. Disease scores were measured at 24 h intervals. Data are presented as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA, Bonferroni post-test. RRV titres in serum, ankle joint and quadriceps of RRV-infected WT and PTX3-/- mice (n = 3–7 per group) at (B) 2 and (C) 10 dpi were determined by plaque assay. Data are presented as mean ± SEM and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, Student unpaired t-test.
Fig 4.
PTX3 modulates expression kinetics of pro-inflammatory mediators during RRV infection in mice.
21-day-old C57BL/6 WT and PTX3-/- (n = 4–7 per group) mice were infected subcutaneously with 104 PFU RRV. Transcriptional profiles of immune mediators, (A) IFN-Ɣ, (B) TNF-α, (C) IL-6 and (D) iNOS were determined by qRT-PCR in the quadriceps at early RRV disease (2 dpi) and peak RRV disease (10 dpi). Data were normalized to HPRT and shown as fold expression relative to WT. Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001, Student unpaired t-test.
Fig 5.
PTX3 delays cellular infiltration kinetics during RRV infection in mice.
(A) 6–7 week old C57BL/6 WT and PTX3-/- (n = 4 per group) mice were infected intraperitoneally with 105 PFU RRV. Peritoneal lavage harvested at 6 hpi was characterized and quantified by flow cytometry using the markers as described in Materials and Methods to determine percentages of neutrophils and inflammatory monocytes. Data are presented as mean ± SEM. ***P < 0.001, Student unpaired t-test. (B) 21-day-old C57BL/6 WT and PTX3-/- (n = 4–7 per group) mice were infected subcutaneously with 104 PFU RRV. Leukocytes were isolated from the quadriceps harvested at 10 dpi. Cells were characterized and quantified by flow cytometry using the markers as described in Materials and Methods. Total numbers of inflammatory monocytes and NK cells are shown. Data are presented as mean ± SEM. *P < 0.05 **P < 0.005, Student unpaired t-test.
Fig 6.
PTX3 enhances RRV replication and viral entry.
(A) HEK293T cells were transfected with human PTX3 or vector plasmid for 20 h before RRV infection at MOI 1 for 24 h. Supernatants were harvested and RRV titres determined by plaque assay. (B) Supernatants of transfected HEK293T cells were harvested at 20 h post transfection and used to treat untransfected HEK 293T cells in the presence of RRV (MOI 1) for 1 h at 37°C, followed by 24 h incubation at 37°C in complete medium. Supernatants were harvested and RRV titres determined by plaque assay. Data are presented as mean ± SEM. *P < 0.05 **P < 0.005, Student unpaired t-test. Transfected HEK293T cells were harvested at 0 and 6 hpi, (C) quantified by flow cytometry using anti-alphavirus antibody for detection of viral entry, and (D) assessed for intracellular PTX3 expression using flow cytometry analysis. Data (n = 3) are presented as mean ± SEM and are representative of 2 independent experiments. ***P < 0.001, two-way ANOVA, Bonferroni post-test. (E) hPTX3-transfected HEK293T cells were fixed at 6 hpi and stained for PTX3 (green) and DAPI. Images are representative of 2 independent experiments. Magnification, ×60. Scale bar, 10 μm.
Fig 7.
PTX3 promotes early viral replication in PTX3-expressing HEK 293T cells co-transfected with RRV.
(A) HEK293T cells were transfected with human PTX3 or vector plasmid for 20 h. Transfected cells were harvested to assess intracellular PTX3 expression using flow cytometry analysis. (B) Vector-/hPTX3-expressing cells were harvested at 20 h post transfection and subjected to a second transfection with RRV through electroporation. Co-transfected cells and supernatant were harvested at 3 and 6 h post RRV transfection and intracellular RRV expression was assessed by flow cytometry using anti-alphavirus antibody. (C) Virus titres in the supernatants were determined by plaque assay. Data (n = 3) are presented as mean ± SEM and are representative of 2 independent experiments. ***P < 0.001, two-way ANOVA, Bonferroni post-test.
Fig 8.
PTX3 enhances RRV replication in murine primary fibroblasts.
(A) Primary tail fibroblasts isolated from WT mice were infected with RRV at MOI 1 for 24 hours. Transcriptional profiles of PTX3 in mock- and RRV-infected fibroblasts were determined by qRT-PCR. Data were normalized to HPRT and shown as fold expression relative to mock-infected cells. (B) Primary tail fibroblasts isolated from WT and PTX3-/- mice were infected with RRV at MOI 1 for 24 hours. Supernatants were harvested and RRV titres determined by plaque assay. (C) Primary tail fibroblasts from PTX3-/- mice were infected with RRV (104 PFU RRV) and pre-bound PTX3-RRV complex (5 μg/ml of mouse recombinant PTX3 + 104 PFU RRV) for 24 hours. Supernatants were harvested and RRV titres determined by plaque assay. (D) Primary tail fibroblasts from WT and PTX3-/- mice were infected with RRV at MOI 1 and harvested at 0 and 6 hpi for viral load analysis to assess viral entry, using viral load qRT-PCR with specific probe and primers against RRV nsP3 RNA, where total RRV nsP3 copy number was calculated and expressed as a percentage relative to WT infected controls, and (E) assessed for intracellular PTX3 expression using flow cytometry analysis. Data (n = 3) are presented as mean ± SEM of percent relative to WT and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, two-way ANOVA, Bonferroni post-test. (F) Primary tail fibroblasts from WT mice were infected with RRV at MOI 1, harvested at 0 and 6 hpi and stained for PTX3 (green) and DAPI (blue). Images are representative of 2 independent experiments. Magnification, ×60. Scale bar, 10 μm.
Fig 9.
PTX3 binds to RRV and colocalizes in the cytoplasm during infection.
(A) Different concentrations of mouse recombinant PTX3 were added to RRV-coated plates for 2 hours at 37°C, followed by binding to biotin-conjugated anti-PTX3 antibody for an additional 2 hours at 37°C. Optical density at 450 nm was read using Horseradish Peroxidase Substrate kit. (B) Vector- and hPTX3-transfected HEK293T cells were fixed at 6 hpi and stained for PTX3 (orange), RRV (magenta) and DAPI. Images are representative of 2 independent experiments. Magnification, ×60. Scale bar, 10 μm.
Fig 10.
N-terminal of PTX3 is essential for binding to RRV and facilitates viral entry.
(A) Schematic representation of structural features of human full-length (FL), N-terminal (N-term) and C-terminal (C-term) PTX3. (B) Different concentrations of human recombinant FL-PTX3, or (C) 5 μg/ml of human recombinant FL-, N-term- and C-term-PTX3, were added to RRV-coated plates for 2 hours at 37°C, followed by binding to biotin-conjugated anti-PTX3 antibody for additional 2 hours at 37°C. Optical density at 450 nm was read using Horseradish Peroxidase Substrate kit. Data are expressed as mean ± SEM of percent binding relative to FL-hPTX3 (n = 4). (D) PTX3-RRV complex-infected HEK293T cells were harvested at 0 and 6 hpi. Virus entry was quantified by flow cytometry using anti-alphavirus antibody. Data (n = 6) are presented as mean ± SEM and are representative of 2 independent experiments. **P < 0.01, ***P < 0.001, one-way ANOVA, Bonferroni’s post-test. (E) HEK293T cells were infected with RRV (104 PFU RRV) and pre-bound PTX3-RRV complex (5 μg/ml of human recombinant FL-, N-term- or C-term-PTX3 + 104 PFU RRV) for 24 hours. Supernatant was harvested and RRV titres was determined by plaque assay on Vero cells.
Fig 11.
Acute phase protein MBL binds to RRV but does not affect viral infectivity.
(A) Serum from RRVD patients (n = 21) or healthy controls (n = 10) were analyzed by ELISA for MBL levels. Data are presented as mean ± SEM. ***P < 0.001, Mann-Whitney U test. (B) 21-day-old C57BL/6 WT mice (n = 4–5 per group) were subcutaneously injected with 104 PFU of RRV or PBS (mock). Mice were sacrificed at 2, 5, 10 and 15 dpi, and serum was collected for analysis of MBL-C expression by ELISA. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, two-way ANOVA, Bonferroni post-test. (C) Increasing concentrations of mouse recombinant MBL-C were added to RRV-coated plates for 2 hours at 37°C, followed by binding to biotin-conjugated anti-MBL-C antibody for additional 2 hours at 37°C. Optical density at 450 nm was read using Horseradish Peroxidase Substrate kit. (D) Dose-dependent infection of C2C12 cells was performed at MOI 0.1, 1, 2.5, 5 and 10 for 24 h, using EFGP-RRV. The percentage of infected cells (EGFP+) was assessed using flow cytometry analysis. (E) C2C12 cells were infected with EGFP-RRV (104 PFU RRV) and pre-bound MBL-C-RRV or PTX3-RRV complex (1 μg/ml of mouse recombinant proteins + 104 PFU RRV) for 6, 12 and 24 hours. The percentage of infected cells (EGFP+) was assessed using flow cytometry analysis. Horizontal dotted line represents the mean percentage of EGFP+ cells detected in mock control. *P < 0.05, ***P < 0.001, one-way ANOVA, Bonferroni’s post-test.