Farnesyltransferase inhibitor FTI-277 inhibits PD-L1 expression on septic spleen lymphocytes and promotes spleen lymphocyte activation

Abstract

Farnesyltransferase inhibitors have been tested in clinical trials for the treatment of tumours. In sepsis, the binding of programmed death 1 (PD-1) to programmed death ligand 1 (PD-L1) promotes lymphocyte apoptosis and decreases cytokine expression, thus affecting survival rates. The PD-1/PD-L1 pathway plays an important role in chronic viral infection, bacterial infection and sepsis. However, the precise immunosuppressive and anti-inflammatory functions of this pathway remain poorly understood. In our previous study, the induction of sepsis by caecal ligation and puncture (CLP) resulted in increased farnesyltransferase activity and farnesylated protein levels in the spleen relative to sham treatment. However, the effect of inhibition of farnesyltransferase activity on overall survival rates in patients with sepsis and the specific signalling pathway involved remain to be investigated. In this study, mice with CLP-induced sepsis were treated with farnesyltransferase inhibitor (FTI-277), and PD-L1 expression on septic spleen lymphocytes was examined. Flow cytometric analysis revealed that PD-L1 is expressed constitutively on lymphocytes and that PD-L1 protein expression was up-regulated strongly following CLP. FTI-277 down-regulated PD-L1 mRNA and protein expression on septic spleen lymphocytes in a dose-dependent manner. This effect was associated closely with nuclear factor kappa B (NF-κB). In addition, the significant damping effect of FTI-277 on the PD-L1 signal promoted interferon (IFN)-γ secretion, interleukin (IL)-2 production and splenocyte proliferation in response to anti-CD3⁺ CD28⁺ antibodies in mice. Furthermore, FTI-277 reduced spleen lymphocyte apoptosis in septic mice. Therefore, FTI-277 regulates spleen lymphocyte activity via the PD-L1 signalling pathway, with significant anti-inflammatory effects attributable to suppression of the NF-κB pathway. Farnesyltransferase represents a valuable therapeutic target for the treatment of sepsis.

Keywords: T cells; inflammation; molecular biology; transcription factors.

Figure 1

Expression of programmed death ligand 1 (PD‐L1) in spleen lymphocytes in sham mice and septic mice. In order to investigate the expression of PD‐L1 in septic spleen lymphocytes, caecal ligation and puncture (CLP) was utilized to induce sepsis in mice. The sham group served as the control. (a) Flow cytometric analysis of spleen lymphocytes for PD‐L1 expression. PD‐L1 was expressed constitutively in the two groups; however, the CLP group exhibited higher PD‐L1 expression than the sham group. (b) Western blotting and (c) reverse transcription–polymerase chain reaction (RT–PCR) analysis indicated the same trends. Data are expressed as mean ± standard deviation (s.d.); *P < 0·05; **P < 0·01. These results are representative of three experiments that yielded similar results. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Farnesyltransferase inhibitor‐277 (FTI‐277) down‐regulates programmed death ligand 1 (PD‐L1) expression and inhibits PD‐L1 mRNA expression on septic spleen lymphocytes. Flow cytometric analysis indicated dose‐dependence of FTI‐277‐induced down‐regulation of PD‐L1 in septic lymphocytes. Following treatment with FTI‐277 (15 mg/kg, 25 mg/kg, 35 mg/kg), PD‐L1 expression was significantly reduced, reaching the lowest levels at 25 mg/kg. (a) The black curve represents the sham group without any stimulation, and the blue curve represents septic mice treated with various concentrations of FTI‐277. (b) Western blotting revealed that FTI‐277 (25 mg/kg) inhibits PD‐L1 protein expression strongly on septic lymphocytes. (c) Reverse transcription–polymerase chain reaction (RT–PCR) indicated that FTI‐277 (25 mg/kg) dramatically decreased the levels of PD‐L1 mRNA in spleen lymphocytes compared with those in animals subjected only to caecal ligation and puncture (CLP). Data are expressed as mean ± standard deviation (s.d.); *P < 0·05; **P < 0·01. These results are representative of three experiments that yielded similar results. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Farnesyltransferase inhibitor‐277 (FTI‐277) inhibits programmed death ligand 1 (PD‐L1) expression on sepsis‐induced spleen lymphocytes and promotes the activation of spleen lymphocytes. Effects of FTI‐277 on spleen lymphocyte activation: sepsis‐induced apoptosis was found to be prevented by FTI‐277 in the mouse spleen. At 16 h after caecal ligation and puncture (CLP), (a) flow cytometric analysis and (b) observation of increased terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL)‐positive apoptotic nuclei indicated that FTI‐277 and PD‐L1 monoclonal antibodies (mAbs) (MIH1) decreased significantly the percentage of apoptotic cells in the spleens of septic mice relative to CLP alone. Effects of FTI‐277 treatment on secretion of (c) interferon (IFN)‐γ and (d) interleukin (IL)‐2: mice were treated with or without FTI‐277 at 2 h after CLP or sham operation. Splenocytes were isolated at 16 h after CLP or sham operation and incubated in a plate coated with anti‐CD3 antibody and soluble anti‐CD28 antibody. IFN‐γ and IL‐2 concentrations in the supernatants were measured by enzyme‐linked immunosorbent assay (ELISA). (e) Effects of FTI‐277 treatment on proliferative response in anti‐CD3CD28 antibody‐stimulated splenocytes. Data are expressed as mean ± standard deviation (s.d.); *P < 0·05; **P < 0·01; ***P < 0·001. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Farnesyltransferase inhibitor‐277 (FTI‐277) down‐regulates programmed death ligand 1 (PD‐L1) expression on septic spleen lymphocytes in a nuclear factor kappa B (NF‐κB)‐dependent manner. FTI‐277 suppresses lipopolysaccharide (LPS)‐induced NF‐κB activity and translocation. Total protein, cytoplasmic protein and nuclear protein were isolated. NF‐κB p65 levels in the (a) cytoplasm and (b) nucleus were analysed by Western blotting. Total protein levels of (c) phosphorylated IκB and (d) NF‐κB p65 were analysed by Western blotting. β‐actin or lamin B was used as a loading control. The data shown are representative of four independent experiments. Data are expressed as mean ± standard deviation (s.d.); *P < 0·05; **P < 0·01; ***P < 0·001.