A novel 1,2-benzenediamine derivative FC-99 suppresses TLR3 expression and ameliorates disease symptoms in a mouse model of sepsis

Abstract

Background and purpose: Sepsis is a clinical condition characterized by overwhelming systemic inflammation with high mortality rate and high prevalence, but effective treatment is still lacking. Toll-like receptor 3 (TLR3) is an endogenous sensor, thought to regulate the amplification of immune response during sepsis. Modulators of TLR3 have an advantage in the treatment of sepsis. Here, we aimed to explore the mechanism of a monosubstituted 1,2-benzenediamine derivative FC-99 {N(1) -[(4-methoxy)methyl]-4-methyl-1,2-benzenediamine}on modulating TLR3 expression and its therapeutic potential on mouse model of sepsis.

Experimental approach: Cells were pretreated with FC-99 followed by poly(I:C) or IFN-α stimulation; TLR3 and other indicators were assayed. Female C57BL/6 mice were subjected to sham or caecal ligation puncture (CLP) surgery after i.p. injection of vehicle or FC-99; serum and tissues were collected for further experiments.

Key results: FC-99 suppressed inflammatory response induced by poly(I:C) with no effect on cell viability or uptake of poly(I:C). FC-99 also inhibited TLR3 expression induced by not only poly(I:C) but also by exogenous IFN-α. This inhibition of FC-99 was related to the poly(I:C)-evoked IRF3/IFN-α/JAK/STAT1 signalling pathway. In CLP-induced model of sepsis, FC-99 administration decreased mice mortality and serum levels of inflammatory factors, attenuated multiple organ dysfunction and enhanced bacterial clearance. Accordingly, systemic and local expression of TLR3 was reduced by FC-99 in vivo.

Conclusion and implications: FC-99 reversed TLR3 expression and ameliorate CLP-induced sepsis in mice. Thus, FC-99 will be a potential therapeutic candidate for sepsis.

Figure 1

The chemical structure of FC-99 and its effect on peritoneal macrophage viability. (A), the chemical structure of FC-99. In (B), peritoneal macrophages were treated with various concentrations of FC-99 for 24 h or (C) with FC-99 at 50 μM for the indicated time periods. Cell viability was measured by the CCK-8 kit assay. Data are means ± SEM. n = 5 in each condition. ***P < 0.001 versus control.

Figure 2

FC-99 inhibited the inflammatory response induced by poly(I:C). Peritoneal macrophages were pretreated with FC-99 for 2 h before 25 μg·mL⁻¹ poly(I:C) stimulation for the indicated time periods; TNF-α and IL-6 levels were measured by Q-PCR (A, B, n = 4) or elisa (C,D, n = 4). (E, F) Protein levels of phospho-ERK, ERK, phospho-JNK, JNK, phospho-P38, P38, phospho-IκB, IκB and GAPDH were measured by Western blot, and a representative of three independent experiments is given. Histograms showed the relative band intensity of Western blot from three independent experiments. The data are means ± SEM. ***P < 0.001 versus control; ^###P < 0.001 versus poly(I:C) treated alone.

Figure 3

FC-99 suppressed TLR3 expression induced by poly(I:C) in vitro. (A) Peritoneal macrophages were pretreated with FC-99 for 2 h and exposed to 25 μg·mL⁻¹ poly(I:C) for the indicated time periods. TLR3 expression was measured by flow cytometry. Shaded grey, isotype control; red line, treated with poly(I:C); blue line, treated with FC-99 and poly(I:C). In (B), the MFI calculated from the histogram. n = 5 in each condition. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Peritoneal macrophages were pretreated with FC-99 for 2 h and exposed to 25 μg·mL⁻¹ poly(I:C) for another 6 h (up, mRNA level) or 24 h (down, protein level) and TLR3 expression in different levels were measured by Q-PCR (n = 4) or Western blot. (D) Histogram shows the relative band intensity of Western blot from three independent experiments. (E) RAW264.7, BM-DCs and splenocytes were pretreated with FC-99 for 2 h and exposed to 25 μg·mL⁻¹ poly(I:C) for another 6 h. TLR3 mRNA levels were measured by Q-PCR (n = 4). Data are means ± SEM. *P < 0.05, ***P < 0.001 versus control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus poly(I:C) treated alone.

Figure 4

TLR3 expression was induced by IFN-α via IFNAR1, whereas FC-99 inhibited the poly(I:C)-induced IFN-α via IRF3. RAW264.7 cells were treated with 25 μg·mL⁻¹ poly(I:C) for the indicated time periods, mRNA levels of IFN-α (A) and TLR3 (B) were measured by Q-PCR (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus control. (C) RAW264.7 cells were transfected with control siRNA (si-NC) or siRNA targeting IFNAR1 (siIFNAR1) for a total of 48 h and IFNAR1 mRNA levels were determined by Q-PCR (n = 4). (D) After transfected for 48 h, cells were treated with 25 μg·mL⁻¹ poly(I:C) for 6 h and TLR3 mRNA were measured by Q-PCR (n = 4). RAW264.7 were pretreated with FC-99 for 2 h before 25 μg·mL⁻¹ poly(I:C) stimulation for the indicated time periods. (E) IFN-α mRNA levels were tested by Q-PCR (n = 4). (F) Protein levels of phospho-IRF3, IRF3 and GAPDH were tested by Western blot. Histogram showed the relative band intensity from three independent experiments. The data are means ± SEM. **P < 0.01, ***P < 0.001.

Figure 5

FC-99 reduced the TLR3 expression induced by exogenous IFN-α via STAT1. RAW264.7 cells (A) or peritoneal macrophage (B) was pretreated with FC-99 for 2 h before IFN-α (100 U·mL⁻¹) stimulation for another 3 h and TLR3 mRNA levels were tested by Q-PCR (n = 4). (C, D) RAW264.7 cells were treated with IFN-α (100 U·mL⁻¹) for the indicated time with or without pretreatment with FC-99 for 2 h; protein levels of phospho-STAT1, STAT1 phospho-JAK1, JAK1 and GAPDH were tested by Western blot. Histograms show the relative band intensity from three independent experiments. The data are means ± SEM. ***P < 0.001 versus control; ^###P < 0.001 versus IFN-α treated alone.

Figure 6

FC-99 inhibited RSV replication in host cells. A549 cells were pretreated with 50 μM FC-99 for 2 h and then infected with RSV (MOI = 1 PFU·per cell) for the indicated time periods. Cells were harvested and fusion proteins F0 and F1 of RSV in host cells were detected by Western blot. Histogram shows the relative band intensity from three independent experiments.

Figure 7

FC-99 improved survival and decreased symptoms of CLP-induced sepsis. Mice were challenged with CLP surgery after i.p. injection of FC-99 (100 mg·kg⁻¹) or vehicle for 2 h. At 24 h after surgery, mice (n = 5 per group) were killed and the serum from each group was collected for biochemical indexes (A) including creatinine, urea nitrogen, ALT and AST, or cytokine/chemokine (C) including IL-6, TNF-α, CCL5 and KC analysis. (B) Lung, liver and kidney sections were subjected to H&E staining. (E) The numbers of bacterial colony forming units (CFU) in peritoneal lavage fluids were quantified. (D) In separate experiments (n = 15 per group), the survival rate was monitored for 6 days. The data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus sham/vehicle group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus CLP/vehicle group.

Figure 8

FC-99 suppressed systemic and local expression of TLR3 in CLP-induced sepsis. Mice were challenged with CLP surgery after i.p. injection of FC-99 (100 mg·kg⁻¹) or vehicle for 2 h and killed at 24 h. (A) Splenic cells were obtained and TLR3 expression was measured by flow cytometry. (B) Histogram shows TLR3 expression in splenic CD11b⁺ cells. (C–E) TLR3 mRNA expression in lung, liver and kidney tissue was quantified by Q-PCR. (F) Schematic representation showing the mechanism of action of FC-99. The data are means ± SEM (n = 5 per group). *P < 0.05, ***P < 0.001 versus sham/vehicle group; ^#P < 0.05, ^###P < 0.001 versus CLP/vehicle group.