Fig 1.
ATX is induced by TLR4 ligand LPS in THP-1 cells dependent on the IRF3-mediated autocrine IFN-β production.
(A) THP-1 cells were stimulated with LPS (0.1 μg/ml) for the indicated times. ATX mRNA expression was detected by qRT-PCR. After LPS stimulation for 24h, the secreted ATX protein in culture medium was detected by Western blot. (B) THP-1 cells were incubated with CHX (1 μM) for 30 min prior to the stimulation with LPS. ATX mRNA expression was analyzed after LPS stimulation for 16h by qRT-PCR. (C) THP-1 cells were transfected with IRF3 siRNA and non-specific siRNA (siNC) respectively. After siRNA transfection for 48 h, THP-1 cells were treated with LPS for 16h. IRF3 protein was detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. (D) THP-1 cells were preincubated for 30 min with IFN-β specific neutralizing antibody (anti-IFN-β; 1 μg/ml) or negative control antibody (rabbit IgG; 1 μg/ml), and then subjected to LPS stimulation. ATX mRNA expression was detected after LPS stimulation for 16h by qRT-PCR. (E) THP-1 cells were transfected with IFNAR1 siRNA and non-specific siRNA (siNC), respectively. After siRNA transfection for 48 h, THP-1 cells were treated with LPS for 16h. IFNAR1 protein was detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 2.
ATX is induced by TLR 9 ligand CpG oligonucleotides (ODN) in THP-1 cells dependent on the IRF7-mediated autocrine IFN-β production.
(A) THP-1 cells were treated with CpG ODN (1 μM) for the indicated times. ATX mRNA expression was detected by qRT-PCR. After CpG ODN treatment for 12h, the secreted ATX protein in culture medium was detected by Western blot. (B) THP-1 cells were transfected with IRF7 siRNA and non-specific siRNA (siNC) respectively. After siRNA transfection for 48 h, THP-1 cells were treated with or without CpG ODN for 6h. IRF7 protein was detected by western blot, and ATX mRNA expression was analyzed qRT-PCR. (C) THP-1 cells were preincubated for 30 min with IFN-α specific neutralizing antibody (anti-IFN-α; 1 μg/ml) or IFN-β specific neutralizing antibody (anti-IFN-β; 1 μg/ml), and then subjected to CpG ODN treatment. ATX mRNA expression was detected after CpG ODN treatment for 6h by qRT-PCR. (D) THP-1 cells were transfected with IFNAR1 siRNA and non-specific siRNA (siNC), respectively. After siRNA transfection for 48 h, THP-1 cells were treated with CpG ODN for 6h. IFNAR1 protein was detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 3.
ATX is induced by TLR 3 ligand poly(I:C) in THP-1 cells dependent on the IRF3-mediated autocrine IFN-β production.
(A) THP-1 cells were treated with poly(I:C) (10 μg/ml) for the indicated times. ATX mRNA expression was detected by qRT-PCR. After poly(I:C) treatment for 12 h, the secreted ATX protein in culture medium was detected by Western blot. (B) THP-1 cells were transfected with IRF3 siRNA and non-specific siRNA (siNC) respectively. After siRNA transfection for 48 h, THP-1 cells were treated with or without poly(I:C) for 6 h. IRF3 protein was detected by Western blot, and ATX mRNA expression was analyzed qRT-PCR. (C) THP-1 cells were preincubated with IFN-α specific neutralizing antibody (anti-IFN-α; 1 μg/ml) or IFN-β specific neutralizing antibody (anti-IFN-β; 1 μg/ml) for 30 min, and then subjected to poly(I:C) treatment. ATX mRNA expression was detected after poly(I:C) treatment for 6 h by qRT-PCR. (D) THP-1 cells were transfected with IFNAR1 siRNA and non-specific siRNA (siNC), respectively. After siRNA transfection for 48 h, THP-1 cells were treated with poly(I:C) for 6 h. IFNAR1 protein was detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 4.
Type I interferons induce ATX expression in THP-1 cells through JAK-STAT and PI3K-AKT pathway.
(A) THP-1 cells were treated with IFN-α (50 ng/ml) or IFN-β (10 ng/ml) for the indicated times. After treatment with IFN-α for 4 h or IFN-β for 6 h, the secreted ATX protein in culture medium was detected by Western blot. (B) THP-1 cells were treated with LY294002 (10 μM), SB202190 (10 μM), PD98059 (20 μM), pyridone 6 (P6; 10 μM) for 30 min before IFN-α or IFN-β treatment. After IFN-α treatment for 2 h or IFN-β treatment for 4 h, ATX mRNA levels were detected by qRT-PCR. (C) THP-1 cells were transfected with AKT siRNA and non-specific siRNA (siNC) respectively. After siRNA transfection for 48 h, THP-1 cells were treated with IFN-α for 2 h or with IFN-β for 4 h. AKT protein was detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. (D) TYK2 and JAK1 siRNAs were transfected into THP-1 cells respectively, with non-specific siRNA (siNC) as the control. After siRNA transfection for 48 h, THP-1 cells were treated with IFN-α for 2 h or with IFN-β for 4 h. TYK2 and JAK1 were detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. (E) STAT1 and STAT3 siRNAs were transfected into THP-1 cells respectively, with non-specific siRNA (siNC) as the control. After siRNA transfection for 48 h, THP-1 cells were treated with IFN-α for 2 h or with IFN-β for 4 h. STAT1 and STAT3 were detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 5.
The synergistic effect of IFN-γ on the ATX induction by IFN-β.
(A) THP-1 cells were treated for 4h with IFN-β (10 ng/ml) and/or IFN-γ (50 ng/ml) as indicated. ATX mRNA levels were detected by qRT-PCR. (B) THP-1 cells were stimulated by LPS for 16 h with or without IFN-γ priming. ATX mRNA levels were detected by qRT-PCR. (C) THP-1 cells were preincubated with IFN-β specific neutralizing antibody (anti-IFN-β; 1μg/ml) or negative control antibody (rabbit IgG; 1 μg/ml) for 30 min, and then subjected to LPS and/or IFN-γ treatment as indicated. ATX mRNA levels were detected by qRT-PCR after treatment for 16 h. (D) IFNAR1 siRNA and non-specific siRNA (siNC) were transfected into THP-1 cells respectively. After siRNA transfection for 48 h, THP-1 cells were treated with LPS plus IFN-γ for 16 h. IFNAR1 were detected by Western blot, and ATX mRNA expression was analyzed by qRT-PCR. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 6.
LysoPLD activity and LPA production were enhanced by IFN-α/β treatment and TLR activation.
THP-1 cells were washed by PBS for three times and cultured with serum-free RPMI 1640, then stimulated by IFN-α (50 ng/ml), IFN-β (10 ng/ml) or LPS (0.1 μg/ml) for 24 h, by CpG ODN (1 μM) or poly(I:C) (10 μg/ml) for 12 h. (A) After stimulation, the lysoPLD activity in concentrated (40-fold) conditioned serum-free culture medium of THP-1 cells was analyzed using FS-3 as substrate. The conditioned serum-free medium of unstimulated THP-1 cells was concentrated (40-fold) and used as controls. (B, C) LPA levels in the supernatant of conditional medium were assayed by mass spectrometry. Data represent the mean and SD of triplicate determinations. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01.
Fig 7.
ATX is induced by LPS and poly(I:C) in moDCs through type I interferon autocrine loop.
(A) TLR3, TLR4 and TLR9 mRNA expression in moDCs were analyzed by RT-PCR. (B) MoDCs were treated with LPS for 1 h or with poly(I:C) for 6h, and then IFN-α and IFN-β mRNA levels were detected by RT-PCR. (C) MoDCs were preincubated with IFN-β specific neutralizing antibody (anti-IFN-β; 1μg/ml) or negative control antibody (rabbit IgG; 1 μg/ml) for 30 min, and then subjected to LPS (0.1 μg/ml) stimulation. ATX mRNA expression was detected by qRT-PCR after LPS stimulation for 16 h. (D) THP-1 cells were preincubated with IFN-α specific neutralizing antibody (anti-IFN-α; 1μg/ml) or IFN-β specific neutralizing antibody (anti-IFN-β; 1μg/ml) for 30 min, and then subjected to poly(I:C) (10 μg/ml) treatment. ATX mRNA expression was detected by qRT-PCR after poly(I:C) treatment for 6 h. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 8.
Type I interferons induce ATX expression in human PBMCs and monocytes.
(A)Human PBMCs and monocytes were treated with IFN-α (50 ng/ml) for 2h or with IFN-β (10 ng/ml) for 4 h, and then ATX mRNA expression was detected by qRT-PCR. (B) HUVECs and Jurkat cells were treated with IFN-α (50 ng/ml) for 2 h or with IFN-β (10 ng/ml) for 4 h, and then ATX mRNA expression was detected by RT-PCR. (C, D)Human PBMCs and monocytes were pretreated with LY294002 (10 μM), SB202190 (10 μM), PD98059 (20 μM), or pyridone 6 (P6; 10 μM) for 30 min before the IFN-α/β treatment. After IFN-α treatment for 2 h or IFN-β treatment for 4 h, ATX mRNA levels were detected by qRT-PCR. The ATX expression detected by qRT-PCR analyses was normalized to expression of GAPDH and presented relative to expression in untreated cells. All qRT-PCR data are expressed as mean values ± SD, n = 3. The p values derived from Student’s t test are (*) p < 0.05, (**) p < 0.01. A representative experiment out of three is shown.
Fig 9.
A proposed model of the ATX induction through type I IFN autocrine and paracrine loops in response to TLR activation.
IFN-α/β was induced during TLR activation through IRF3/7, and then IFN-α/β functions as autocrine and paracrine factor to induced ATX expression in immune cells through the IFNAR-mediated JAK-STAT and PI3K-AKT pathways. The induction of ATX enhances LPA production in cellular microenvironment and activates the LPA-LPA receptor signaling to modulate immune responses.