A Novel Fungal Immunomodulatory Protein (PCP) Isolated from Poria cocos Induces Toll-like Receptor 4-dependent Activation in Mouse Peritoneal Macrophages and Promotes T Cell Activation and Th1 Immune Response

• Main Authors: Hui-Hsin Chang, 張慧欣
• Other Authors: 許輔
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/56283350608241697595

博士 === 國立臺灣大學 === 園藝學研究所 === 97 === Poria cocos (Schw.) Wolf is an important Oriental medical fungus with multiple functionalities, yet its bioactive substances and mechanisms involved have not been fully characterized. The objective of the present study was to investigate the bioactive substance from P. cocos and its molecular mechanism involved in immune modulation focused on macrophage and lymphocyte activation. A novel immunomodulatory protein (P. cocos immunomodulatory protein; PCP) was purified from the dried sclerotium of P. cocos (Schw.) Wolf using DE-52 cellulose and gel filtration chromatography. Chromatography and electrophoresis results indicated that the native PCP (35.6 kDa) is a disulfide-linked heterodimeric glycoprotein consisting of 14.3 and 21.3 kDa subunits with N- and O-glycosylation. PCP was capable of stimulating RAW 264.7 macrophages in vitro through the induction of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) as well as the regulation of nuclear factor-kappa B (NF-κB)-related gene expression. In primary mouse macrophages, we observed an increase in the expression of major histocompatibility complex (MHC) class II and CD86 molecules on peritoneal cavity macrophages. PCP directly activated macrophages to induce Toll-like receptor (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent signaling. We demonstrated the cell surface interactions of PCP with TLR4 and the capacity of PCP for TLR4 tyrosine phosphorylation. Results obtained with peritoneal macrophages from TLR4-deficient C57BL/10ScN mice revealed that PCP-induced activation and PCP cell surface binding were significantly attenuated. Moreover, enzymatic deglycosylation decreased PCP-mediated responses, indicating that the glycosylated portion of PCP was a key factor in PCP signaling through TLR4 in peritoneal macrophages. Further investigation on lymphocyte activation indicated that PCP directly activated mouse splenocytes, markedly increased cell proliferation and the levels of interferon-gamma (IFN-γ) secretion but not IL-5 production. Similarly, the selectively enhanced transcriptional expression of IL-2 and IFN-γ by PCP was demonstrated using quantitative real-time PCR. Although there were slight increases in the total cell population of CD4+ and CD8+ T cells in PCP-stimulated splenocytes, PCP significantly increased expression of the activation marker CD69 on both splenic CD4+ and CD8+ T cells. The potent CD4+ and CD8+ T cell-activating capability of PCP was demonstrated by the enhancement of cell proliferation, cytokine secretion, activation marker CD44 and CD69 expression upon anti-CD3/CD28 costimulation. The expression of T-bet, tyrosine phosphorylation of STAT4, IFN-γ and IL-2 secretion during PCP-induced CD4+ T cell activation were upregulated. In contrast to the functional deficiency of deglycosylated PCP on macrophage activation, the core protein of PCP was shown to be involved strongly in induction of T cell activation, as demonstrated by inhibition of T cell response using deproteinized PCP. In vivo experiments indicated that oral administration of PCP (50 mg/kg body weight) suppressed the level of serum IgG1, and enhanced amounts of serum IgG2a and T helper 1 (Th1)-associated cytokine secretion in BALB/c mouse spleen cell cultures. Oral administration of PCP upon immunization with ovalbumin (OVA) exhibited that OVA specific IgG2a levels were also significantly increased compared with those of PBS-treated mice, suggesting that PCP could suppressed OVA-induced Th2 response to drive Th1 development. Taken together, these studies characterize a new potential immune stimulator, PCP, which induces TLR4-dependent activation within murine macrophages and triggers a Th1-dominant immune response. These observations provide strong support for further studies of PCP and P. cocos to explore their overall modulatory nature toward mammalian cells and reveal their pharmaceutical potential and industrial value.