Fig 1.
MAYV infection trigger caspase-1 activation in macrophages.
(A-F) PAM(3)CSK(4)-primed bone marrow-derived macrophages (BMDMs) were treated with mock or infected with Zika virus (ZIKV), chikungunya virus (CHIKV) or Mayaro virus (MAYV) at a MOI of 0.2, 1 or 5. After 24 hours of infection, the levels of IL-1β (A-C) and LDH (D-F) in the cell-free supernatants were measured. (G-I) BMDMs were infected with MAYV at a MOI of 5 (MOCK were used as control) for 24 hours infection and the cells were stained for active CASP1 (using FAM-YVAD) and analyzed as shown by the representative contour plots (G). The percentage (H) and integrated mean of fluorescence (iMFI) (I) of activated cells is shown. (J) After 24 hours of infection, supernatants (SN) and cellular extracts (CE) were harvested from MOCK or MAYV-infected BMDMs, and levels of cleaved CASP1 (p20) and IL-1β (p17) were detected by western blotting the SN and probing for the indicated proteins. As loading controls, levels of pro-caspase-1 and β-actin were also assessed in the CE. Data are shown as means ± standard deviation (SD) of triplicate samples (A-I) and are representative of three (A-J) independent experiments that yielded similar results. Statistical analysis was performed by student’s t test. *, P < 0.05.
Fig 2.
The NLRP3 inflammasome is activated in macrophages infected with MAYV.
(A-D) WT bone marrow-derived macrophages (BMDMs) were infected with Mayaro virus (MAYV) at an MOI of 5 (MOCK was used as a control), or treated with RPMI medium (NI) or ultrapure LPS (500 ng mL-1) as negatives and positive controls, respectively. After 3 and 6 hours of infection, cells were lysed and the RNA was extracted for qPCR analysis of Casp1 (A), Nlrp3 (B), Aim2 (C) and Asc (D). (E) PAM(3)CSK(4)-primed BMDMs derived from WT, Nlrp3–/–, Asc–/–, Casp1/11–/–and Aim2–/–mice were infected with MAYV at a MOI of 5. After 24 hours of infection, cell-free supernatants were harvested and IL-1β was quantified by ELISA. (F) Primed WT and Nlrp3–/–BMDMs were infected with either fresh MAYV, heat inactivated (HI) or UV-inactivated (UVI) virus. After 24 hours, the levels of IL-1β were measured by ELISA. (G) Western Blotting was performed in WT BMDMs after 24 hours of infection. Supernatants (SN) and cellular extracts (CE) were harvested from MAYV-infected (or MOCK) BMDMs, and levels of cleaved caspase-1 (p20) and IL-1β (p17) were detected in the SN. As loading controls, levels of pro-caspase-1 and β-actin were assessed in the CE. (H-J) WT and Nlrp3–/–BMDMs were infected with MAYV at a MOI of 5. After 24 hours of infection the cells were stained for active-caspase-1 with FAM-YVAD and analyzed as shown by the representative histograms (G). The percentage (H) and integrated mean of fluorescence (iMFI) (I) of activated cells is shown. Data shown are means ± SD of triplicate samples (A-E, G-I) and are representative of the data obtained from two (A-D, F) or three (E, G-H) independent experiments. Statistical analysis was performed by student’s t test. *, P < 0.05.
Fig 3.
Potassium efflux, ROS and cathepsin B are required for efficient inflammasome activation.
(A-D) WT bone marrow-derived macrophages (BMDMs) were infected with Mayaro virus (MAYV) at a MOI of 5 or MOCK infected for 90 minutes or treated with PMA or Rotenone (positive controls). Cells were stained for 30 minutes with, the fluorescent dyes H2DCFDA and MITOSOX, which stain total and mitochondrial ROS, respectively, harvested and analyzed by FACS. Representative histograms (A, C) and integrated mean of fluorescence (iMFI) data for each dye are shown (B, D). (E) After 2 hours of infection, BMDMs were incubated with APG-2 dye and the levels of intracellular potassium were determined as described in the methods. (F-I) Cells were left untreated or treated with apocynin (50 or 100 μM) for 1 hour and then infected with MAYV or treated with PMA (positive control). ROS production (F) was evaluated after treatment, and IL-1β levels (G) were assessed after 24 hours of infection by ELISA. BMDMs were primed with PAM(3)CSK(4) and incubated for 3 hours prior to infection with the indicated concentrations of NaCl (H) and KCl (I). After 24 hours, cell-free supernatants were collected and IL-1β levels were measured by ELISA. (J-O) MAYV RNA levels was determined by qPCR in Apocynin, KCl and NaCl-treated BMDMs after 1 (J-L) or 24 hours (M-O) of infection. Data shown are mean ± SD of quadruplicate samples and are representative of three independent experiments performed. Statistical analysis was performed by student’s t test. *, P < 0.05.
Fig 4.
A mouse model to study acute inflammation by MAYV in vivo.
WT mice (n = 5 per injected group) were injected subcutaneously into the footpad with 10 μL of conditioned media (mock), MAYV (106 PFU) or CHIKV (107 PFU). (A) Footpad thickness was measured daily (Footpad height) through the 8th day of infection, and (B) representative images at day 6 after infection are shown. (C) The vonFrey test was used to measure pain in the paws of mock- and MAYV-injected mice. (D) Mice were euthanized at 1, 3, 6 and 10 days after infection and the amount of MAYV RNA was quantified in the footpad, muscle and spleen. (E) Histological examination (hematoxylin and eosin) of footpad sections from control (MOCK) or MAYV-infected mice after 6 days. Ke: keratin; Ep: epidermal layer; De: dermal layer; Mus: muscle; Od: oedema; V: Vessel; PMN: polymorphonuclear cell infiltration. Scale bars = 10 μM.
Fig 5.
NLRP3 signaling participates in the pathogenesis of MAYV infection in vivo.
WT and Nlrp3–/–mice (n = 5 per injected group) were injected subcutaneously into the footpad with 10 μL of MAYV (105 or 106 PFU) or mock infected. Footpad thickness height (H) X width (W) was measured daily through to the 10th day of infection in mock (A) and MAYV-injected mice with 105 PFU dose (B) or 106 PFU dose (C). Representative images of paws at day 6 after infection are shown (D). WT, Nlrp3–/–(Genentech), Nlrp3–/–(Jackson), Caspase1/11-/- mice (n = 5 per injected group) were injected subcutaneously into the footpad with 10 μL of MAYV (105 PFU) or mock infected and footpad thickness was measured daily through to the 10th day of infection (E). The von-Frey test was used to measure pain in the paws of mock (F) and MAYV-injected mice (G). (H-J) WT and Nlrp3–/–mice were euthanized at 1 and 6 days after infection and the amount of MAYV RNA was quantified in the footpad (H), muscle (I) and spleen (J). Virus titers were determined in the same organs by plaque assay (K) and are represented as mean ± SD. WT, Nlrp3–/–and Casp1/11-/- mice were injected with Mock or MAYV for 5 days. Footpads were obtained, and both IL-1β (L) and IL-18 (M) were quantified in the homogenate’s supernatants. Data shown are representative of three (A-D) or two (E-J) independent experiments. Statistical analysis was performed by two-way ANOVA with Bonferroni’s multiple comparison test (M). Asterisks indicate significant differences (P < 0.05) between WT and Nlrp3–/–—infected groups, while in 5E asterisks indicate significant differences (P < 0.05) between WT and immune deficient-infected groups.
Fig 6.
NLRP3 contributes to cell recruitment and tissue damage upon MAYV infection.
WT and Nlrp3–/–mice (n = 3 per injected group) were injected subcutaneously into the footpad with 10 μL of MAYV (106 PFU) or Mock. (A) Histological analysis of the mouse footpad histology sections after inoculation with Mock or MAYV. Inoculated feet were dissected, processed for histological analysis and stained with H&E. In MAYV-infected images, muscle necrosis and inflammation are shown by asterisks (*), whereas “#” labels regions of edema and inflammation of the subcutaneous layer and “o” points to muscle inflammation. Images are representative of at least 20 fields of view. Scale bars: 100 μM (Left panels); 20 μM (Right panels). (B, C) The number of neutrophils (B) and the tissue damage score (C) was quantified in the footpads of these mice. (D) Articular lavage was obtained from the knees and total cell numbers were quantified by Cytospin. Statistical analysis was performed by student’s t test (M). Data are representative of two independent experiments, and asterisks indicate significant differences (P < 0.05) between WT and Nlrp3–/–-infected groups.
Fig 7.
NLRP3 plays an important role in the recruitment of myeloid cells to the infected tissue.
WT, Nlrp3–/–and Casp1/11–/–mice (n = 4 to 6 mice per injected group) were injected subcutaneously into the footpad with 10 μL of Mock, MAYV or CHIKV (106 PFU). After 5 days of infection, footpads were removed and single cell suspensions were obtained for FACS analysis. (A) Representative contour plots showing the frequency of neutrophils (CD11b+Ly6G+) and inflammatory monocytes (CD11b+Ly6Chigh) in each experimental group. Frequencies of both types of cells (B,D) and their absolute numbers (C,E) are shown. Data are representative of two independent experiments. Statistical analysis was performed by student’s t test. Asterisks indicate significant differences (P < 0.05) between MAYV-infected Nlrp3-/- or Casp1/11-/- compared to WT mice, whereas # indicate differences (P < 0.05) between CHIKV-infected Nlrp3-/- or Casp1/11-/- compared to WT mice (C,E).
Fig 8.
The inflammasome limits infiltration of NK cells into mice footpads.
WT, Nlrp3–/–and Casp1/11–/–mice (n = 4 to 6 mice per injected group) were injected subcutaneously into the footpad with 10 μL of Mock, MAYV or CHIKV (106 PFU). After 5 days of infection, footpads were removed and single cell suspensions were obtained for FACS analysis. (A) Representative contour plots showing the frequency of NK cells (CD45+NK1.1+CD3-) in each experimental group. Graphical quantification of the percentage of NK cells (B) and its absolute numbers (C) are shown. (D-I) T (CD45+CD3+NK1.1-), NKT cells (CD45+CD3+NK1.1+) and B cells (CD45+CD19+) were assessed in injected footpads. Frequencies of these types of cells (D,F,H) and their absolute numbers (E,G,I) are shown. Data are representative of two independent experiments. Statistical analysis was performed by student’s t test (M). Asterisks indicate significant differences (P < 0.05) between MAYV-infected Nlrp3-/- or Casp1/11-/- compared to WT mice, whereas # indicate differences (P < 0.05) between CHIKV-infected Nlrp3-/- or Casp1/11-/- compared to WT mice (B,D).
Fig 9.
Caspase-1 p20, IL-1β and IL-18 are elevated in the serum of MAYV-infected patients in comparison to healthy individuals.
Serum samples of 13 MAYV-infected patients and 19 healthy control samples were included. Samples were diluted 1:2 for caspase-1 p20 ELISA (A), 1:5 for IL-1β ELISA (B) and 1:3 for IL-18 ELISA (C). Data are represented as scatterplots showing means ± SEM of samples. Statistical analysis was performed by student’s t test. Asterisks indicate significant differences (P < 0.05) between MAYV-infected and control groups.