Fig 1.
F. nucleatum induces proinflammatory cytokines production in Caco-2 Cells.
(A and B) Caco-2 cells were infected by F. nucleatum for the indicated periods of time (A; MOI = 100:1) or at different MOIs (B; 10, 50, 100 and 200) for 12 h. Supernatants were assessed by ELISA for levels of IL-8, IL-1β and TNF-α. (C) Representative images of hematoxylin and eosin staining of colonic epithelium from C57BL/6 mice. Data are presented as the means ±SEM of three experiments. *P<0.05.
Fig 2.
Inflammation induced by F. nucleatum is dependent on ROS.
(A) Caco-2 cells were infected by F. nucleatum for the indicated periods of time (3, 6, 12, 24 hours). ROS generation was detected by DCFH-DA assay. (B, C and D) Following pretreatment with 1 mM Tiron or 10 mM N-acetyl-cysteine (NAC) for 6 hours, Caco-2 cells were infected with F. nucleatum (MOI = 100:1) for 12 hours. Supernatants of medium was assessed by ELISA for levels of IL-8, IL-1β and TNF-α. The data are presented as the means ±SEM of at least 3 independent experiments. *, P<0.05.
Fig 3.
Autophagy is impaired in Caco-2 cells after F. nucleatum infection.
(A) Caco-2 cells were transfected with a GFP-MAP1LC3B plasmid for 24 hours, and then infected with F. nucleatum for 12 hours. Confocal microscopy determined the number of GFP-MAP1LC3B puncta in each cell. (B) The protein level of SQSTM1 and the rate of MAP1LC3B-II to β-actin in Caco-2 cells were detected with western blot assay after F. nucleatum infection 12 hrs. (C) Caco-2 cells were infected with F. nucleatum for 12 hrs in the presence of Baf-A1 (10 nM), and western blot assay detected SQSTM1 and MAP1LC3B-II. Data are presented as the means ±SEM of three experiments. *P<0.05.
Fig 4.
Inhibition of autophagy enhances cytokines production and ROS generation induced by F. nucleatum infection.
(A, B, and C) After pretreatment of 0.1% DMSO, 3-MA (2mM), Baf-A1 (10 nM) or Rapa (100 nM), Caco-2 cells were infected with F. nucleatum (MOI = 100:1) for 12 hrs. Supernatants of medium were assessed by ELISA for levels of IL-8, IL-1β and TNF-α. (D, E and F) Production of IL-8, IL-1β and TNF-α in Caco-2 cells transfected with siRNA specific for ATG5 or ATG12 (50 nM) for 24h and infected with F. nucleatum (MOI = 100) for 12 hrs, as assessed by ELISA. (G) siRNAs targeting ATG5 or ATG12 (100 nM each) transfected Caco-2 cells for 24 hrs, and the protein levels were assayed by Western blotting. (H and I) After pretreatment of 0.1% DMSO, 3-MA (2mM), Baf-A1 (10 nM) or Rapa (100 nM), or transfected with siRNA specific for ATG5 or ATG12 (50 nM) for 24h, Caco-2 cells were infected with F. nucleatum (MOI = 100:1) for 12 hrs. ROS generation was detected by DCFH-DA assay. The data shown are the means ±SEM of three experiments. *, P<0.05.
Fig 5.
Schematic of the proposed mechanism of Fusobacterium nucleatum-induced impairment of autophagic flux enhancing the expression of proinflammatory cytokines via ROS in Caco-2 Cells (see text for details).