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

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.

Figure 1. Purification and biochemical characterization of YZP.

A. Fruiting bodies of Trametes versicolor (Yun-Zhi). B. Chromatogram of the crude protein extract of T. versicolor fractionated on a HiTrap Q anion exchange column. C. Chromatogram of the pooled YZP-containing fractions fractionated on a Resource Q anion exchange column. D. Chromatogram of the purified YZP samples analyzed by PROTEIN KW-802.5 SHODEX gel-filtration chromatography. E. SDS-PAGE analysis of the crude protein extracts from T. versicolor (lanes 1 and 5), the YZP-containing fraction (lane 2), non-YZP fraction (lane 3), and purified YZP (lanes 4 and 6). Lanes 1 to 4 were stained with CBR, whereas lanes 5 and 6 were stained with PAS reagent. Lane M was loaded with PageRuler Prestained Protein Ladder #SM0671. F. The calibration curve was established via linear regression of the molecular weights of the proteins present in the PageRuler Prestained Protein Ladder #SM0671 versus the nature logarithm of their relative mobility shifts. The molecular weights of T. versicolor proteins are indicated with red arrows.

Figure 2. Complete nucleotide and amino acid sequences of YZP.

RNA-seq and de novo contig assembly were conducted to identify the YZP gene. The complete nucleotide sequence of the YZP cDNA was confirmed by PCR cloning using the primers shown in Supporting Information Table S1 and Table S2, and is presented in plain capital letters with the start and termination codons presented in red. The nucleotide sequences of one set of cloning primers (YZP1) are indicated with blue arrows. The translated amino acid sequence is presented in bold capital letters, whereas the N-terminal amino acids are underlined. The putative signal peptide predicted using the SignalP 4.0 Server (Supporting Information Figure S3) is indicated with a grey background.

Figure 3. YZP activates plasma cell differentiation and IgM production in mice B cells.

A. The purity of the MACS-purified CD19⁺ B cells was examined by analyzing the expression of CD3 and B220 via flow cytometry. B. The MACS-purified CD19⁺ B cells were treated with YZP (20 μg/mL) for 24 h, and the cell size and granularity was examined by analyzing the forward scatter (FSC) and side scatter (SSC) via flow cytometry. C. MACS-purified CD19⁺ B cells were treated with YZP (20 μg/mL) for 24 h, followed by analysis of the expression levels of the indicated surface markers by flow cytometry. The shaded gray color indicates untreated B cells, whereas the solid line indicates B cells treated with YZP. The numbers indicate the geometric mean fluorescence intensity. D. and E. MACS-purified CD19⁺ B cells were treated with YZP (20 μg/mL) for the indicated periods, and subsequently the expression level of CD138 was analyzed via flow cytometry. The numbers indicate the percentage of gated CD138^high cells.

Figure 4. YZP induces IL-6 and IL-10 production in mice B cells.

A. MACS-purified CD19⁺ B cells were treated with the indicated dosages of YZP for 72 h, and the culture supernatant was then collected for IgM measurements. B. MACS-purified CD19⁺ B cells were treated with YZP (10 μg/mL) for 72 h, and subsequently the culture supernatant was collected for IgM and IgG measurements. C. and D. MACS-purified CD19⁺ B cells were treated with the indicated dosages of YZP for 72 h, and subsequently the culture supernatant was collected for cytokine measurements.

Figure 5. YZP stimulates CD1d⁺ B cells to produce IL-10 and increases CD1d expression in mice B cells.

A. Mice splenocytes were treated with YZP (20 μg/mL) for 48 h, and subsequently the cells were harvested for PerCP-Cy5.5-labeled anti-mouse CD3 and FITC-labeled anti-mouse B220 surface staining and PE-labeled IL-10 intracellular staining. B. MACS-purified CD19⁺ B cells were stimulated with YZP (20 μg/mL) for 48 h, and subsequently the cells were harvested for PerCP-Cy5.5-labeled anti-mouse CD5 and FITC-labeled anti-mouse CD1d surface staining and PE-labeled IL-10 intracellular staining. C. The CD1d expression of B cells treated with YZP (20 μg/mL) or untreated for 48 h. The numbers indicate the percentage of gated CD1d⁺ cells.

Figure 6. YZP-induced B cells inhibit inflammatory cytokine release from LPS-activated macrophages.

A. Mice peritoneal macrophages (2×10⁶) were stimulated with or without LPS (1 μg/mL) for 12 h and subsequently co-cultured with or without an equal number (2×10⁶) of YZP-induced B cells or control B cells for 12 h. The culture supernatant was subsequently collected for TNF-α and IL-1β measurements. B. Mice peritoneal macrophages (2×10⁶) were stimulated with LPS (1 μg/mL) for 12 h and were subsequently co-cultured with the indicated numbers of YZP-induced B cells for 12 h. The culture supernatant was collected for TNF-α and IL-1β measurements. C. and D. Mice peritoneal macrophages (1×10⁶) were stimulated with or without LPS (1 μg/mL) for 12 h and subsequently co-cultured with or without 2×10⁶ YZP-induced B cells or control B cells for 12 h. The culture supernatant was subsequently collected for TNF-α and IL-1β measurements. The data is presented as the mean ± SD, where the asterisks indicate the statistical differences (*, p<0.05; **, p<0.01; N.S., non-significant) among groups.

Figure 7. YZP-induced B cells alleviate disease activity in colitic mice.

A. C57BL/6J mice were injected i.p. with PBS (0.2 mL; •), YZP-induced B cells (1.5×10⁷ in 0.2 mL PBS; ▴), or control B cells (1.5×10⁷ in 0.2 mL PBS; Δ) 1 day before 3% DSS induction. One group of control mice was administered PBS i.p., and the remaining mice ingested normal drinking water (○). The body weights of the mice were monitored daily, and the data are presented as the mean ± SD, where the asterisks indicate significant differences among DSS-ingested groups (p<0.05, n = 5). B. The disease activity index was scored based on the degree of weight loss, stool consistency, and fecal bleeding as detailed in Supporting Information Table S3 and was monitored daily. The data are presented as the mean ± SEM, and the asterisks indicate significant differences among DSS-ingested groups (p<0.05, n = 5).

Figure 8. YZP-induced B cells ameliorate colonic damage in colitic mice.

A. The colon lengths of healthy mice (•) and DSS-ingested mice that received i.p. PBS (▪), YZP-induced B cells (B_YZP; ▴), or control B cells (B_CTR; ▾) were measured. One representative colon sample from each group is shown in the right panel. The asterisk indicates significant differences among DSS-ingested groups (p<0.05, n = 5). B. Two colon samples from each group were fixed in 10% buffered formalin, and 5-μm-thick sections were cut and stained with H&E. H&E-stained cross-sections were examined with a microscope. One representative colon section is shown. The blue arrowheads indicate intact goblet cells, and the red arrowheads indicate infiltrated leukocytes. C. Total RNA was extracted from 3 colon samples from each group and reverse transcribed into cDNA for cytokine gene expression analysis. The data are presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) between DSS-ingested, PBS i.p. mice, and B_YZP i.p. mice. The # symbols indicate significant differences (p<0.05) between DSS-ingested, B_CTR i.p. mice, and B_YZP i.p. mice.

Figure 9. 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).

Figure 10. Blockages of both TLR2 and TLR4 were required to inhibit B cell differentiation induced through YZP.

A. MACS-purified TLR2^–/– and TLR4^–/– CD19⁺ B cells were pre-incubated with isotype-matched IgG (30 μg/mL), anti-TLR2 mAb (30 μg/mL), or anti-TLR4 mAb (30 μg/mL) for 1 h and 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 then harvested for CFSE staining cell proliferation analysis. B. The culture supernatant was collected, and the levels of secreted IgM and (C and D) IL-10 were measured. The data are presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) among YZP-treated groups.

Figure 11. YZP binds to B cells through TLR2 and TLR4 and activates the gene expression of TLR-signaling molecules.

A. MACS-purified CD19⁺ B cells from WT, TLR2^–/–, and TLR4^–/– mice were pre-incubated with YZP (shaded grey), FITC-labeled YZP, or FITC-labeled BSA for 30 min. The cells were subsequently harvested for flow cytometry analysis. The numbers indicate the percentages of positive fluorescent cells among the total cells. The right panel shows the results from 3 independent experiments, presented as the mean ± SD (n = 3). The asterisks indicate significant differences (p<0.05) between TLR^–/– and WT mice. B. MACS-purified CD19⁺ B cells were stimulated with YZP (10 μg/mL) for the indicated periods. Total RNA was extracted and subsequently reverse transcribed to cDNA for gene expression analysis. The asterisks indicate that the data are significantly different (p<0.05) from the 0 h control group.