Immunomodulatory Effects Of Polysaccharopeptide (psp) In Human PBMC Through Regulation Of TRAF6/TLR Immunosignal-transduction Pathways

Objective: The Coriolus Versicolor ( C. versicolor ) is a macrofungi belonging to the Polyporaceae family of fungi. Polysaccharopeptide ( PSP ) was extracted from C. versicolor, and has been proved to be a valuable adjuvant for the combination with chemotherapy or radiotherapy in the treatment of various cancers. To understand the mechanism of PSP on the immunomodulation, we examined cytokine secretion associated with immunosignal-transduction signaling in human peripheral blood monouclear cells ( PBMCs ). And then we investigated the mRNA expression levels and / or protein levels of the key nodes of the Toll-Like Receptor ( TLR ) signal transduction pathway.Methods: PBMCs were isolated by Ficoll-Hypaque density centrifugation. PBMCs were then treated with Phytohemagglutinin ( PHA, final concentration 100μg/mL ) in control group or treated with PHA ( final concentration 100μg/mL ) and PSP ( final concentration 25μg/mL ) in experimental group. Cytokine antibody array were used to identify differential cytokines secretion of PBMCs in the presence or absence of PSP. The expression of the key genes and proteins related to TLR signaling and its downstream pathway was determined by RT-PCR or Western blot, such as TIR-containing adaptor molecule ( TRIF ),TRIF-related adaptor molecule ( TRAM ),TNF-receptor associated factor ( TRAF6 ),Myeloid different factor 88 ( MyD88 ),MyD88 adaptor-like protein ( MAL) and nuclear factorκB ( NF-κB ) and so on.Results: Compared with the control group, five of cytokines ( G-CSF, GM-CSF, IL-1α, IL-6, IFN-γ) were up-regulated more than 1.3 times in PBMCs under PSP treatments. Three cytokines were slightly decreased, including IL-2, IL-3 and IL-5. The levels of other cytokines had no significant effects. The mRNA levels of TRAM, TRAF6 ( P < 0.05) and NF-κB ( P < 0.01 ) , which are the key molecules of TLR signaling pathway, were markedly increased. The mRNA levels of TRIF were slightly increased, while the mRNA levels of MAL and MyD88 were slightly decreased. Moreover, the protein level of TRAF6, TRAM and NF-κB were also markedly increased ( P < 0.01 ), the protein level of MyD88 were markedly decreased ( P < 0.01 ).Conclusions: PSP regulated cytokine secretion related to TLR signaling pathway in human PBMCs. TLR signal transduction pathway could be divided into two sub-pathways including MyD88 dependent and TRIF dependent ( MyD88-independent ). The two sub-pathways are involved in immune signal transduction. Especially TRAM-TRIF-TRAF6 sub-signaling pathway of TLR may be one of the key associated signaling pathways in the immunomodulation of PSP.