Page 61 - M. Immunology

Inotodiol, a novel lanostane triterpenoid from Chaga mushroom Inonotus Obliquus induces
characteristic maturation of dendritic cells
Perry Ayn Mayson A. Maza , Ji-Hyun Lee , Minsook Ryu , Ludmila P. Ponomarenko , Valentin A. Stonik , Jong-Yong Kwak 1,2,3
1,2
1,2
4
4
3
1 Department of Pharmacology, School of Medicine, Ajou University Suwon 16499, South Korea, 2 Department of Biomedical Sciences, Graduate school, Ajou University Suwon 16499, South Korea ,
3 Department of Allergy, Ajou University School of Medicine, Suwon 16499, South Korea, 4 G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of
Sciences, 690022, Vladivostok, Russian Federation
BACKGROUND AIM
Inonotus obliquus (I. obliquus), also known as Chaga mushroom, is a rich source of Immature DCs become mature effector DCs that promote the development of TH1,
natural compounds which offer a wide range of biologically active components. TH2 or regulatory T cells (Kapsenberg, 2003). However, the immature DCs alone
Based from the chemical analysis of I. obliqus, polysaccharides, triterpenes and and DCs without co-stimulatory signal and cytokine production can induce T cell
polyphenols are responsible for most of its therapeutic effects. One of these anergy. In addition, DCs in semi-mature state lack certain phenotypic markers or
components is inotodiol, a lanostane-type triterpenoid that specifically has various produce lower amounts of pro-inflammatory cytokines, which can lead to tolerogenic
biological activities, including anti-tumor, anti-viral, and anti-inflammation. Recently, outcome after interaction with responding T cells. Thus, cytokine production of
inotodiol was shown to ameliorate allergy symptoms through acting on masts cells activated DCs is important in generating effector responses in T cells. In this study,
but not on CD4+ T cells in a mouse model of food allergy. However, to our we aim to elucidate the immunological capabilities of inotodiol on bone-marrow
knowledge, there are no data available on the immunomodulatory capacity of derived dendritic cells (BMDCs) and T cell Proliferation.
inotodiol, and no information on DC modulation by its reported immunomodulatory
capabilities.
METHODS

RESULTS
Purification of inotodiol Increased expression of MHC-II, CD86, and Lanosterol also failed to up-regulate Inotodiol upregulates expression of MHC-II
CD40 on BMDCs in in vitro and in vivo the expression of CD86, MHC-II, and and CD86 without secretion of inflammatory
cultures CD40 in BMDCs cytokines

Fig. 1. Purified inotodiol from I. obliquus. (A) Fig. 2. Expression levels of MHC-II and costimulatory Fig. 3. Effects of lanosterol, inotodiol, and Fig. 4. Effects of inotodiol on cytokine production from BMDCs
Chemical structure of lanosterol, inotodiol and molecules in inotodiol-treated BMDCs. (A) CD86 lanosteral on CD86, MHC-II and CD40 and macrophages. (A) cytokine array of supernatants from
lanosteral. (B) HPLC analysis to show the purity expression in inotodiol-treated BMDCs. (B) Dose- expression in BMDCs. inotodiol-treated peritoneal macrophages. (B) The production of
of inotodiol sample. (C) 1 H NMR spectrum to dependent increase of CD86 expression in BMDCs by TNF-α and IL-6 from inotodiol- and LPS-treated BMDCs and
detect chemical structure of inotodiol sample. inotodiol. (C) Expression of MHC-II, CD80 and CD40 in Raw263.7 cells. Supernatants were collected to detect the
inotodiol-treated BMDCs. (D) Expression of CD86 in secretions of IL-12p40, IL-10, and TNF-α by ELISA.
splenic CD11c + DCs after injection of inotodiol to mice.
T-cell proliferation by Inotodiol-BMDCs Inotodiol induced activation of DC without Effects of AKT and NF-κB inhibitors Inotodiol activates the PI-3 kinase/AKT
secretion of inflammatory cytokines and on the expression of CD86 in pathway, but not the p38 MAPK and NF-
Inotodiol-BMDCs increased T cell proliferation Inotodiol-BMDCs kB pathway in DCs
through production of IL-2 alone

Fig. 5. Effects of inotodiol-treated BMDCs on T cell Fig. 6. Cytokine production in coculture of inotodiol-treated Fig. 7. Effects of kinase inhibitors on the expression of Fig. 8. Phosphorylation of kinases in inotodiol-treated BMDCs
proliferation. Inotodiol-treated and OVA-pulsed BMDCs and T cells. Inotodiol-treated and OVA-pulsed CD86 in indotodiol-treated BMDCs.
inotodiol-treated BMDCs were co-cultured with inotodiol-treated BMDCs were cocultured with splenic T
CFSE-labeled splenic T cells (A) and OT-I CD8 + T cells (MLR) and OT-I CD8+ T cells (OT-I) for 4 days,
cells (B) for 4 days, respectively. respectively as in Fig 6.
CONCLUSION
Inotodiol increased the expression of surface maturation markers, including MHC-II, CD86, and CD40 on BMDCs with almost no production of various cytokines, including
TNF-α and IL-12p40 from the BMDCs. OT-I Tcells primed by OVA-pulsed inotodiol-treated BMDCs proliferated and produced IL-2 with production of other cytokines,
including IL-12p40 and IFN-γ. Specific inhibitor of PI3K abrogated the effects of inotodiol on phosphorylation of AKT and upregulation of CD86 and MHC-II. Inotodiol failed to
induce phosphorylation of IKK and degradation of IkB-α and increased expression of CD86 by inotodiol was not blocked by NF-kB inhibitor. These results suggest that
inotodiol induces DCs a characteristic type of maturation through PI3K activation in independence of NF-kB and inotodiol-treated DCs enhance T cell proliferation by
production of IL-2.
REFERENCES: Blagodatski A: Medicinal mushrooms as an attractive new source of natural compounds for future cancer therapy. Oncotarget 49: 29259-29274;
Kapsenberg M, Dendritic-Cell Control of Pathogen-Driven T-Cell Polarization, Nature reviews Immunology; Merad Miriam, Dendritic Cells: controllers of adaptive immunity,
Nature reviews Immunology