Mincle and human B cell function

Abstract

C-type lectin receptors are pattern recognition receptors that are critical for autoimmunity and the immune response. Mincle is a C-type lectin receptor expressed by a variety of antigen presenting cells including macrophages, neutrophils, dendritic cells and B cells; a variety of stimuli including stress are known to induce the expression of Mincle. Mincle is an FcRγ-associated activation receptor that senses damaged cells and upon ligation induces activated macrophages to produce inflammatory cytokines. Recently, while several studies have reported that Mincle plays an important role in macrophage responses to fungal infection its function on B cells remains to be defined. In efforts to elucidate the function of Mincle expressed by B cells, we studied the expression of Mincle on subsets of B cells and analyzed cytokines and synthesized immunoglobulin upon ligation of Mincle. The expression of Mincle on CD27-CD19(+) naïve B cells is significantly higher than CD27 + CD19(+) memory B cells. The stimulation of TLR9 ligand induced Mincle expression on B cells. Furthermore, co-stimulation of TLR9 and Mincle ligand reduced IgG and IgA production from B cells without a significant change in the inflammatory cytokines TNF-α, IL-6, IL-8 and IL-10. Our data identifies Mincle as a potentially critical player in human B cell responses.

Figure 1

Mincle expression on B cells was determined using standard flow cytometry. (A) Representative profiles of Mincle and isotype expression on normal human B cells were displayed. (B) Analysis of the frequency of Mincle positive cells and MFI value of Mincle on CD19⁺ B cells. The frequency of Mincle positive cells was significantly increased (Left panel). The MFI value of Mincle was significantly increased (Right panel). (n=23) (C) Naïve and memory B cells were determined by CD27 antibody. The frequency of Mincle positive CD27−CD19⁺ naïve B cells was significantly higher than CD27+CD19⁺ memory B cells (Left panel). The MFI value of Mincle on CD27−CD19⁺ naïve B cells was significantly higher than CD27+CD19⁺ memory B cells (Right panel). (n=8) * p<0.05, ****p<0.0001

Figure 2

Analysis of expression level of Mincle on CD19⁺ B cells in PBMC’s cultured in the presence of LPS or CpG-B for 4 days (n=15). (A) The frequencies of CD19⁺ B cells in culture of PBMC with either medium control, LPS or CpG were not difference. (B) The frequency of Mincle positive CD19⁺ B cells in PBMC’s cultured in the presence of CpG was significantly higher than medium control (Left panel). The MFI value of Mincle on CD19⁺ B cells in PBMC’s cultured in the presence of CpG was significantly higher than medium control (Right panel). The frequency and MFI of Mincle positive CD19+ B cells in PBMC’s cultured in medium control and the presence of LPS were not statistically difference. **p<0.01

Figure 3

Comparison of expression level of Mincle on CD19+ B cells in PBMC’s cultured in the presence of 100 ng of TDB, 0.5 µM of CpG-B, 100 ng of TDB + 0.5 µM of CpG-B, or media alone for 4 days (n=8). (A) The frequencies of Mincle positive CD19⁺ B cells in PBMC’s cultured in the presence of TDB, CpG or CpG+TDB were significantly higher than medium control. In addition, the frequencies of Mincle positive CD19⁺ B cells in PBMC’s cultured in the presence of CpG or CpG+TDB were significantly higher than TDB. (B) The MFI values of Mincle positive CD19⁺ B cells in PBMC’s cultured in the presence of TDB, CpG or CpG+TDB were significantly higher than medium control. The MFI values of Mincle were not different between TDB, CpG-B and CpG-B+TDB co-stimulation. * p<0.05, **p<0.01

Figure 4

Analysis of immunoglobulin production in supernatant of PBMC or isolated CD19⁺ B cells cultured in the presence of 100 ng of TDB, 0.5 µM of CpG-B, 100 ng of TDB + 0.5 µM of CpG-B, or media alone for 4 days (n=8). Left panel is cultured PBMC, and right panel is cultured CD19⁺ B cell. O.D. 450nm value was determined using ELISA. IgA and IgM levels in supernatants of PBMC’s cultured in media containing CpG were significantly higher than medium control. IgG levels in supernatant of PBMC’s cultured in media containing TDB were significant lower than medium control. IgG and IgA levels in supernatants of PBMC cultured in media containing CpG + TDB were significantly lower than CpG, although IgM level was not different (Left panel). IgG, IgA and IgM levels in supernatants of CD19⁺ B cells cultured in media containing CpG were significantly higher than medium control. IgG and IgA levels in supernatants of CD19⁺ B cells cultured in media containing CpG + TDB were significantly lower than CpG, although IgM level was not different (Right panel). *p<0.05, **p<0.01

Figure 5

Analysis of inflammatory cytokine production from CD19⁺ B cells cultured in the presence of 100 ng of TDB, 0.5µM of CpG-B, 100 ng of TDB + 0.5µM of CpG-B and media alone for 4 days (n=8). TNF-α, IL-10, IL-6 and IL-8 are increased after CpG-B stimulation. Inflammatory cytokines are no difference between CpG-B stimulation and CpG-B +TDB co-stimulation. **p<0.01