(5R)-5-hydroxytriptolide inhibits IFN-gamma-related signaling.
OBJECTIVE
(5R)-5-hydroxytriptolide (LLDT-8) displayed anti-arthritis and anti-allogenic transplantation rejection activities in our previous studies. Here, we aim to further clarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-gamma.


METHODS
T cells were activated with anti-CD3 antibody or concanavalin A (ConA). The expression of cell surface molecules was detected with flow cytometry. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to test cell division. IFN-gamma production was determined by enzyme-linked immunosorbent assay. Cell proliferation was evaluated by [3H]-thymidine uptake. Mice were immunized with ovalbumin to assess the in vivo immune response. RT-PCR and Real-time PCR were applied to determine the mRNA expression. The protein phosphorylation levels were detected by Western immunoblot assay.


RESULTS
LLDT-8 at 100 nmol/L did not change the CD25, CD69, and CD154 expressions in anti-CD3-stimulated T cells. LLDT-8 markedly blocked the cell division of CD4 and CD8 T cells after ConA stimulation. LLDT-8 inhibited T cell-derived IFN-gamma production. Moreover, LLDT-8 suppressed the ovalbumin-specific T cell proliferation and IFN-gamma generation. In anti-CD3-activated T cells, LLDT-8 abrogated the mRNA expression of signal transducer and activator of transcription1 (STAT1), T-box transcription factor, IL-12 receptor beta2, STAT4, and interferon regulatory factor 1 in the IFN-gamma expression pathway. Western blot analysis showed that LLDT-8 blocked the phosphorylation levels of extracellular signal-regulated kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28-activated T cells. In addition, LLDT-8 reduced the transcripts of macrophage inflammatory protein (Mip)-1alpha, Mip-1beta, regulated upon activation normally T-cell expressed and secreted, inducible protein-10, IFN-inducible T cell a chemoattractant, and monokine induced by IFN-gamma in IFN-gamma-stimulated murine macrophage cell line Raw 264.7 cells.


CONCLUSIONS
LLDT-8 was a potential inhibitor for IFN-gamma-associated signaling.
Zhou
Ru
R
Laboratory of Immunopharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Wang
Jun-xia
JX
Tang
Wei
W
He
Pei-lan
PL
Yang
Yi-fu
YF
Li
Yuan-chao
YC
Li
Xiao-yu
XY
Zuo
Jian-ping
JP
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D004224:Diterpenes / Q000494:pharmacology*; D005260:Female; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D050794:STAT1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000096:biosynthesis / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics
C509074:5-hydroxytriptolide; D004224:Diterpenes; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; D020825:T-Box Domain Proteins; D007371:Interferon-gamma; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases