Reishi Polysaccharides Induce Immunoglobulin Production through the TLR4/TLR2-mediated Induction of Transcription Factor Blimp-1

Kuo-I Lin,Yeong-Yi Kao,Hui-Kai Kuo,Wen-Bin Yang,Alice Chou,Hsin-Hung Lin,Alice L Yu,Chi-Huey Wong

doi:10.1074/jbc.m601106200

Kuo-I Lin, Yeong-Yi Kao + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m601106200

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Abstract

The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.

Highlights

The crude or purified components of Reishi extracts possess anti-tumor and immunomodulating activities [1,2,3]
As previously described [9], we observed that Reishi-F3 treatment results in B cell activation as shown by the increased expression of the activation marker, CD86, on surfaces and the increased proliferation of murine splenic B cells after 3 days of treatment
NMR analysis revealed differential signature of components between LPS and Reishi-F3.3 These results suggest that Reishi-F3 may have a role in promoting plasmacytic differentiation

Summary

EXPERIMENTAL PROCEDURES

Reishi-F3 Preparation—The preparation of the polysaccharide- containing F3 fraction of G. lucidum (Reishi-F3) was performed essentially as previously described [4, 25]. Purified splenic B cells (purity Ͼ95%) were cultured at RPMI 1640 medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin/streptomycin (100 units/ml), 2 mM L-glutamine, and 50 ␮M 2-mercaptoethanol at the density of 2 ϫ 106 cells/ml. Enriched human B cells (purity Ͼ90%) were maintained in RPMI 1640 medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin/streptomycin (100 units/ml), and 2 mM L-glutamine. The following procedures and methods for detecting captured mouse IgM, human IgM, or human IgG were essentially according to the manufacturer’s suggestions (Bethyl Laboratories), using TMB as the substrate. For the detection of multiple cytokines from Reishi-F3 treated human peripheral B cells, we essentially followed the protocol provided by the manufacturer (Upstate Cell Signaling Solutions). DNA oligonucleotides containing Blimp-1 binding sites were synthesized and labeled with biotin using the LightShift chemiluminescent EMSA kit (Pierce) following the manufacturer’s provided protocol. The oligonucleotides corresponding to various Blimp-1 binding sites or control in this study were as follows: PRF, 5Ј-CGCGTACAGAAAGGGAAAGGACTAGCGCG-3Ј; CD86, 5Ј-AAATAATTAGAAAGAGAAAACAAACCTC-3Ј; nonspecific, 5Ј-AGCTTTAGCCGCAATATGCCGATATCC-3Ј

RESULTS

DISCUSSION

Ig induction