Fig 1.
Stat1-/- mice display increased production and proteolytic maturation of IL-1β upon gastrointestinal MNV infection.
Age- and sex-matched Stat1+/- and Stat1-/- littermates were infected with 10/ PFU of UV-inactivated (UV) or live MNV via oral gavage. At 3 days post-infection ileum, Peyer`s patches (PP), mesenteric lymph nodes (MLN), spleen and liver lysates were prepared for Multiplex ELISA (A, B) and Western Blotting analyses (C-E). (A) Heat maps showing relative expression of chemokines and cytokines as measured by Multiplex ELISA in the indicated tissues of UV- or Live MNV infected Stat1+/- and Stat1-/-mice. Data shown are the means of Stat1+/-UV-MNV mice (n = 7), Stat1+/-Live-MNV mice (n = 8), Stat1-/-UV-MNV mice (n = 5) and Stat1-/-Live-MNV mice (n = 8), normalized per mean log2 expression. (B) Raw data from heat map analysis showing IL-1β levels in indicated tissues. Samples indicated in blue and red were analyzed by (C-E) Western blotting for proteolytic maturation of IL-1β in (C) MLN, (D) spleen and (E) liver. Statistics: Log-linear regression analysis, p values in (A, B) indicate association with Stat1-/-Live-MNV set-up, with *p<0.05; **p<0.01; ***p<0.001.
Fig 2.
MNV induces secretion of mature IL-1β by activating the caspase-1- and ASC-dependent Nlrp3 inflammasome.
Bone-marrow-derived macrophages (BMDMs) from (A-C) WT mice; (D-F) WT and Caspase-11-/- mice; (G-I) WT, Caspase-1/11-/- and ASC-/- mice; or (J-L) WT and Nlrp3-/- mice were left untreated (unprimed) or were TLR2-primed after which they were mock infected (control) or infected with either UV-inactivated or live MNV, both at a MOI 5. Cell culture supernatants collected after indicated time periods (A) or after 24h (D, G, J) were analyzed for secreted IL-1β by ELISA; and (B, C, E, F, H, I, K, L) cell lysates prepared 24h post-infection was immunoblotted for IL-1β and caspase-1 maturation. Data shown in (A, D, G, J) are the means ± SD of triplicate wells from a representative experiment out of at least two independent experiments. Data shown in (B, C, E, F, H, I, K, L) are representative for at least two independent experiments.
Fig 3.
Nlrp3-dependent GSDMD cleavage mediates pyroptosis in MNV-infected macrophages.
(A-C) Unprimed WT bone-marrow-derived macrophages (BMDMs) were infected with either UV-inactivated or live MNV at a MOI 5. (A) Sytox Green (SG) uptake and Caspase-3/-7 (C3/7) activity were assessed for 24 hours by IncuCyte real-time monitoring every 30 minutes; (B) confocal microscopic picture of Propidium Iodide uptake and cell morphology at 13 hours post-infection. White arrows indicate cells displaying swollen necrotic morphology; (C) cell lysates were prepared at indicated time points post-infection and were immunoblotted for GSDMD and caspase-3 processing. (D-I) BMDMs from WT and indicated KO mice were (D) left untreated (unprimed) or were (E-I) TLR2-primed, after which they were infected with either UV-inactivated or live MNV at a MOI 5. (D, E) Sytox Green (SG) uptake was assessed for 24 hours by IncuCyte real-time monitoring every 30 minutes. (F-H) Cell lysates were prepared at 24 hours post-infection and were immunoblotted for (F-G) GSDMD processing along with (H) IL-1β and caspase-1 maturation. (I) Cell culture supernatants collected at indicated time points post-infection were analyzed for secreted IL-1β. Data shown in (A, D-E) are the means of duplicate wells from a representative experiment out of two independent experiments; data shown in (B-C, F-H) are representative for at least two independent experiments; data shown in (I) are the means ± SD of triplicate wells from a representative experiment out of two independent experiments.
Fig 4.
STAT1 suppresses the secretion of mature IL-1β from unprimed MNV-infected macrophages through inhibiting MAVS-mediated pro-IL-1β induction.
(A-C) Unprimed bone-marrow-derived macrophages (BMDM) from Stat1+/- and Stat1-/- littermates were mock infected (control) or infected with either UV-inactivated or live MNV, both at a MOI 5. After 24 hours (A) culture supernatants were analyzed for secreted IL-1β by ELISA; (B) cell lysates were immunoblotted for IL-1β, caspase-1, and GSDMD processing; and (C) at indicated time points MNV genome copy numbers in BMDMs were determined. (D-E) Unprimed bone-marrow-derived macrophages (BMDM) from Stat1+/-, Stat1-/- and Stat1-/-Mavs-/- mice were mock infected (control) or infected with either UV-inactivated or live MNV, both at a MOI 5. After 24 hours (D) supernatant was analyzed for secreted IL-1β levels by ELISA; and (E) cell lysates were immunoblotted for IL-1β and caspase-1 processing. Data shown in (A, D) are the means ± SD of triplicate wells from a representative experiment out of two independent experiments. Data shown in (B, E) are representative for 2 independent experiments. Data shown in (D) represent means ± SD of BMDMs derived from three mice per genotype, analyzed in three independent experiments, each with triplicate wells.
Fig 5.
Nlrp3 as well as GSDMD contribute to MNV-induced lethality and intestinal inflammation in Stat1-/- mice.
(A, B) Unprimed BMDMs from Stat1-/-Nlrp3+/- and Stat1-/-Nlrp3-/- mice were left untreated (Control) or infected with either UV-inactivated or live MNV at MOI 5. 24 hours post-infection (A) culture supernatant was analyzed for secreted IL-1β and (B) cell lysates were immunoblotted for IL-1β, caspase-1 and GSDMD maturation. (C) Age- and sex-matched Stat1-/-Nlrp3+/- and Stat1-/-Nlrp3-/- littermates were infected with 10/ PFU MNV via oral gavage. Four days post-infection (C) spleen and (D) MLN lysates were prepared and were immunoblotted for proteolytic maturation of (C-D) IL-1β and (D) GSDMD. Each lane represents a different mouse. (E) Kaplan-Meier survival curve of Stat1-/-Nlrp3+/- and Stat1-/-Nlrp3-/- littermates infected with 106 PFU of MNV via oral gavage. (F) Kaplan-Meier survival curve, (G) fecal consistency and (H) fecal Lipocalin-2 levels at indicated days post-infection of age- and sex-matched Stat1-/-, Stat1-/-Nlrp3-/- and Stat1-/-Gsdmd-/- mice infected with 106 PFU of MNV via oral gavage. Six Stat1-/- mice from (F) were unable to produce feces at three days post-infection and were therefore omitted from the analyses in (G) and (H). Data shown in (A) are the means ± SD of triplicate wells from a representative experiment out of 2 independent experiments. Data shown in (B) is representative for 2 independent experiments. Data shown in (E-H) represent graphs each combined from 2 independent experiments. Statistics (E, F) Nonparametric log-rank test; (G, H) Mann Whitney U-test; ns not significant; *p<0.05; **p<0.01; ***p<0.001.