Table 1.
Gender, age, blood IL-6 and MFHAS1 concentrations in septic patients and controls.
Fig 1.
The expression of MFHAS1 in PBMC and macrophages/monocytes after TLR2 stimulation.
(A) PBMC was isolated from the peripheral blood of humane in the control group and the septic patients. The gene expression of MFHAS1 in the PMBC was analyzed by qPCR. n = 8/group. (B) After stimulation with 10 ng/mL Pam3CSK4 for designated time, RAW 264.7 macrophages indicated a time lag in Mfhas1 expression. The expressions of MFHAS1 and GAPDH were detected by western blotting. (C) The corresponding optical density of MFHAS1 bands normalized with GAPDH. (D) After stimulation with 10ng/mL Pam3CSK4 for designated time, THP-1 indicated a time lag in MFHAS1 expression. The expressions of MFHAS1 and GAPDH were detected by western blotting. (E) The corresponding optical density of MFHAS1 bands normalized with GAPDH. Data are presented as mean ± SD in each group. Image J was used in optical density measurement otherwise as indicated. *p < 0.05, compared with control.
Fig 2.
MFHAS1 inhibits the transcriptional activity of NF-κB and IL-6 production 6 h after stimulation with Pam3CSK4 through TLR2.
(A) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, and 100 ng NF-κB-dependent luciferase reporter plasmid as well as 10 ng renilla plasmid. Post transfection for 24 h, these transfected cells were exposed to mock treatment, Pam3CSK4 100 ng/mL or 10 μg/mL for 6 h, and fold increase in luciferase activity was measured for NF-κB activation using dual luciferase kits. The relative luciferase activity was calculated from the ratio of NF-κB-Luc (firefly) activity to renilla activity. (B) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2, and 24 h post-transfected cells were untreated or exposed to Pam3CSK4 100 ng/mL or 10μg/mL. After treatment for 6 h, IL-6 expression was assayed by quantitative RT-PCR and normalized to GAPDH. (C) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2, and 24 h post-transfected cells were untreated or exposed to Pam3CSK4 100 ng/mL. The cell supernatant was collected and the amounts of IL-6 were determined by ELISA at 6 h post-treatment. Values are the means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, or ***p < 0.001.
Fig 3.
MFHAS1 activates NF-κB, AP-1, and IL-6 expression 24 h after stimulation with Pam3CSK4 through TLR2.
(A, B) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, an100 ng NF-κB luciferase reporter plasmid (A) or 20 ng AP-1 luciferase reporter plasmid (B) and 10 ng renilla plasmid. 24 h post-transfected cells were exposed to mock treatment, Pam3CSK4 100 ng/mL or 10μg/mL. At 24 h posttreatment, fold increase in luciferase activity was measured for NF-κB or AP-1 activation using dual luciferase kits. The relative luciferase activity was calculated from the ratio of NF-κB/AP-1 (firefly) activity to renilla activity. (C, D) HEK 293 cells and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, and 24 h post-transfected cells were untreated or exposed to Pam3CSK4 100 ng/mL. After 24 h and 36 h posttreatment, induction of IL-6 expression was assayed by quantitative RT-PCR and normalized to β-actin (C). Cell supernatant was collected and the amounts of IL-6 were determined by ELISA (D). Values are the means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, or ***p < 0.001.
Fig 4.
IL-6 and TNF-α production after stimulation with Pam3CSK4 by knockdown of Mfhas1 using shMFHAS1.
(A, B) RAW 264.7 macrophages were transfected with shMFHAS1 or pCDH empty vector or HIS-MFHAS1 plasmid, and 24 h post-transfected cells were treated with 10 ng/mL Pam3CSK4. Cell supernatant was collected after 6, 12, and 24 h posttreatment, and IL-6 (A) and TNF-α (B) production were measured by ELISA. (C, D) RAW 264.7 macrophages were transfected with shMFHAS1 or pCDH empty vector, and 24 h post-transfected cells were untreated or treated with 10 ng/mL Pam3CSK4 (C), or 50 ng/mL LPS (D). At 6 h and 24 h posttreatment, IL-6 expression was measured by RT-PCR and normalized to β-actin. (E, F) The knockdown efficiency of shMFHAS1 at 6 h and 24 h was assessed by RT-PCR (E) and western blotting (F). Values are the means ± SD from at least three independent experiments. *P < 0.05.
Fig 5.
MFHAS1 does not affect transcription factor IRF-7 and IFN-β expression.
(A) HEK293 and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 or 100 ng TLR2/50 ng CD14 expression plasmids, the pFR luciferase reporter gene along with plasmid expressing IRF7-Gal4 3 ng. Then these 24 h post-transfected cells were treated with mock, 100 ng/mL or 10μg/mL Pam3CSK4 for 24 h, and luciferase reporter gene activity was measured. (B) HEK293 and 293-MFHAS1 cells were transiently transfected with 100 ng TLR2 expression plasmids, and 24 h post-transfected cells were untreated or treated with 100 ng/mL Pam3CSK4 for 24 h. IFN-β mRNA expression was assayed by quantitative RT-PCR. (C) The relative IFN-β mRNA level was normalized to GAPDH. Values are the means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, or ***p < 0.001.
Fig 6.
MFHAS1 induces phosphorylation of JNK and p38.
(A) RAW 264.7 macrophages were transfected with HIS-MFHAS1 or pCDH empty vector plasmid, and 24 h post-transfected cells were treated with 10 ng/mL Pam3CSK4 for 0, 1, 3, 5, 30, 60, 90 min, respectively. Cells were collected and the protein levels of pJNK, JNK, pp38, p38, pERK, ERK, HIS-MFHAS1 and GAPDH were examined by western blotting. (B) Quantified data of the pJNK, pp38, pERK levels. Levels of pJNK, pp38, pERK respectively normalized to JNK, p38, ERK levels were shown.
Fig 7.
MFHAS1 activates pJNK in a time-dependent manner.
(A) RAW 264.7 macrophages were transfected with HIS-MFHAS1 or pCDH empty vector plasmid, and 24 h post-transfected cells were untreated or treated with 10 ng/mL Pam3CSK4 for 6 h or 24 h. Cells were collected and pJNK, JNK, p-p65, GAPDH and HIS-MFHAS1 protein levels were determined by western blotting. (B) Quantified data of the pJNK and p-p65 levels. Levels of pJNK normalized to JNK levels, and pJNK/p-p65 normalized to GAPDH were shown.
Fig 8.
Model of the effect of MFHAS1 on TLR2 signaling pathway in response to TLR2 stimulation for 6–24 h.