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Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

Kuan YC, Wu YJ, Hung CL, Sheu F - PLoS ONE (2013)

Bottom Line: YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d.Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene.

View Article: PubMed Central - PubMed

Affiliation: Center for Biotechnology, National Taiwan University, Taipei, Taiwan ; Department of Horticulture, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.

Results: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.

Conclusions: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

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YZP-induced B cell activation is dependent on TLR2 and TLR4.A. MACS-purified CD19+ B cells were pre-incubated with isotype-matched IgG (30 or 60 μg/mL), anti-TLR2 mAb (30 μg/mL), anti-TLR4 mAb (30 μg/mL), or anti-TLR2 mAb plus anti-TLR4 mAb (30 μg/mL each) for 1 h. The cells were subsequently stimulated with YZP (10 μg/mL) for 48 h. One group was left untreated as a negative control, and one group was stimulated with YZP without antibody pre-incubation as a positive control. The cells were subsequently harvested for intracellular IL-10 staining. The numbers indicate the percentages of IL-10-positive cells among the total cells. B. The culture supernatant was collected for IL-10 and IgM quantification. The data are presented as the mean ± SD (n = 3). The asterisks indicate significant differences among groups (*, p<0.05;**, p<0.01; ***, p<0.005).
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pone-0072422-g009: YZP-induced B cell activation is dependent on TLR2 and TLR4.A. MACS-purified CD19+ B cells were pre-incubated with isotype-matched IgG (30 or 60 μg/mL), anti-TLR2 mAb (30 μg/mL), anti-TLR4 mAb (30 μg/mL), or anti-TLR2 mAb plus anti-TLR4 mAb (30 μg/mL each) for 1 h. The cells were subsequently stimulated with YZP (10 μg/mL) for 48 h. One group was left untreated as a negative control, and one group was stimulated with YZP without antibody pre-incubation as a positive control. The cells were subsequently harvested for intracellular IL-10 staining. The numbers indicate the percentages of IL-10-positive cells among the total cells. B. The culture supernatant was collected for IL-10 and IgM quantification. The data are presented as the mean ± SD (n = 3). The asterisks indicate significant differences among groups (*, p<0.05;**, p<0.01; ***, p<0.005).

Mentions: Fungal proteins have been shown to modulate immune responses through TLR2 and/or TLR4 ligation [14], [29], [30], [32]. In addition, signal transduction mediated through TLRs is required for the development and function of Breg [33]. The use of neutralizing Abs to block TLR2 and/or TLR4 inhibited the ability of YZP to induce IL-10+ Bregs (Figure 9A); TLR4 neutralization caused approximately 40% of the reduction in IL-10 and IgM production, while TLR2 neutralization caused a 14% reduction in IL-10 production. When both TLR2 and TLR4 were blocked through Abs, the YZP-induced IL-10+ Breg population and secreted IL-10 and IgM were reduced by 41%, 22%, and 33%, respectively (Figure 9B). We used gene knockout mice to determine if the loss of the individual TLR2 or TLR4 genes abrogates the effects of YZP on B lymphocytes. B cells purified from TLR2–/– and TLR4–/– mice proliferated and secreted IgM in response to YZP (Figure S4). The application of TLR4- and TLR2-neutralizing Abs completely inhibited YZP function in TLR2–/– and TLR4–/– B cells, respectively (Figure 10). These results indicated that interactions with TLR2 and TLR4 might be crucial for the B cell-activating effect of YZP.


Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

Kuan YC, Wu YJ, Hung CL, Sheu F - PLoS ONE (2013)

YZP-induced B cell activation is dependent on TLR2 and TLR4.A. MACS-purified CD19+ B cells were pre-incubated with isotype-matched IgG (30 or 60 μg/mL), anti-TLR2 mAb (30 μg/mL), anti-TLR4 mAb (30 μg/mL), or anti-TLR2 mAb plus anti-TLR4 mAb (30 μg/mL each) for 1 h. The cells were subsequently stimulated with YZP (10 μg/mL) for 48 h. One group was left untreated as a negative control, and one group was stimulated with YZP without antibody pre-incubation as a positive control. The cells were subsequently harvested for intracellular IL-10 staining. The numbers indicate the percentages of IL-10-positive cells among the total cells. B. The culture supernatant was collected for IL-10 and IgM quantification. The data are presented as the mean ± SD (n = 3). The asterisks indicate significant differences among groups (*, p<0.05;**, p<0.01; ***, p<0.005).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760908&req=5

pone-0072422-g009: YZP-induced B cell activation is dependent on TLR2 and TLR4.A. MACS-purified CD19+ B cells were pre-incubated with isotype-matched IgG (30 or 60 μg/mL), anti-TLR2 mAb (30 μg/mL), anti-TLR4 mAb (30 μg/mL), or anti-TLR2 mAb plus anti-TLR4 mAb (30 μg/mL each) for 1 h. The cells were subsequently stimulated with YZP (10 μg/mL) for 48 h. One group was left untreated as a negative control, and one group was stimulated with YZP without antibody pre-incubation as a positive control. The cells were subsequently harvested for intracellular IL-10 staining. The numbers indicate the percentages of IL-10-positive cells among the total cells. B. The culture supernatant was collected for IL-10 and IgM quantification. The data are presented as the mean ± SD (n = 3). The asterisks indicate significant differences among groups (*, p<0.05;**, p<0.01; ***, p<0.005).
Mentions: Fungal proteins have been shown to modulate immune responses through TLR2 and/or TLR4 ligation [14], [29], [30], [32]. In addition, signal transduction mediated through TLRs is required for the development and function of Breg [33]. The use of neutralizing Abs to block TLR2 and/or TLR4 inhibited the ability of YZP to induce IL-10+ Bregs (Figure 9A); TLR4 neutralization caused approximately 40% of the reduction in IL-10 and IgM production, while TLR2 neutralization caused a 14% reduction in IL-10 production. When both TLR2 and TLR4 were blocked through Abs, the YZP-induced IL-10+ Breg population and secreted IL-10 and IgM were reduced by 41%, 22%, and 33%, respectively (Figure 9B). We used gene knockout mice to determine if the loss of the individual TLR2 or TLR4 genes abrogates the effects of YZP on B lymphocytes. B cells purified from TLR2–/– and TLR4–/– mice proliferated and secreted IgM in response to YZP (Figure S4). The application of TLR4- and TLR2-neutralizing Abs completely inhibited YZP function in TLR2–/– and TLR4–/– B cells, respectively (Figure 10). These results indicated that interactions with TLR2 and TLR4 might be crucial for the B cell-activating effect of YZP.

Bottom Line: YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d.Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene.

View Article: PubMed Central - PubMed

Affiliation: Center for Biotechnology, National Taiwan University, Taipei, Taiwan ; Department of Horticulture, National Taiwan University, Taipei, Taiwan.

ABSTRACT

Background: Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized.

Results: We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway.

Conclusions: We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

Show MeSH
Related in: MedlinePlus