Micro RNA-98 suppresses interleukin-10 in peripheral B cells in patient post-cardio transplantation

Abstract

The immune tolerance to the transplant heart survival is critical. Regulatory B cells are one of the major immune regulatory cell populations in the immune tolerance. Micro RNAs (miR) can regulate the activities of immune cells, such as the expression of interleukin (IL)-10 by B cells. This study tests a hypothesis that micro RNA (miR)-98 plays a role in the regulation of interleukin (IL)-10 expression in B cells (B10 cell) after heart transplantation. In this study, the peripheral blood samples were collected from patients before and after heart transplantation. The expression of miR-98 and IL-10 in B cells was assessed by real time RT-PCR. An allograft heart transplantation mouse model was developed. We observed that after heart transplantation, the frequency of peripheral B10 cell and the IL-10 mRNA levels in peripheral B cells were significantly decreased, the levels of miR-98 were increased in peripheral B cells and the serum levels of cortisol were increased in the patients. Treating naive B cells with cortisol in the culture suppressed the expression of IL-10 in B cells, which was abolished by knocking down the miR-98 gene. Administration with anti-miR-98, or cortisol inhibitor, or adoptive transfer with B10 cells, significantly enhanced the survival rate and time of mice received allograft heart transplantation. In conclusion, the enhancement of serum cortisol affects the immune tolerant feature of B cells, which can be attenuated by anti-miR-98-carrying liposomes.

Keywords: B lymphocyte; cortisol; heart transplantation; interleukin-10; micro RNA.

Figure 1. Assessment of peripheral B10 cells

(A–C) the gated dot plots show the frequency of B10 cells in peripheral blood mononuclear cells of healthy subjects and patients one week before and one week after heart transplantation. (D) the isotype IgG staining results; used as a negative control. (E) the scatter dot plots show the data of A–C. The data were from 20 healthy subjects and 20 patients. Samples from individual patients were processed and analyzed separately. Data of bars are presented as mean ± SD. *p < 0.01, compared with the healthy subjects.

Figure 2. Assessment of “stress hormones” in patients after heart transplantation

The bars indicate the urinary levels of cortisol (A), serum cortisol levels (B), urinary noradrenaline (C) and urinary adrenaline (D) in 20 healthy subjects, 20 patients before and after heart transplantation. Samples from individual patients were processed and analyzed separately. Data of bars are presented as mean ± SD. *p < 0.01, compared with the healthy subjects. E, the correlation between peripheral B10 cells and urinary cortisol levels (samples were collected one week before surgery and one week after surgery).

Figure 3. Assessment of miR-98 in B cells

(A) the peripheral B cells were isolated from the blood samples collected from 20 healthy subjects and 20 patients a week before surgery and 3 days after surgery. The bars indicate the miR-98 levels in peripheral B cells of healthy subjects and patients. (B) the positive correlation between serum cortisol levels and the miR-98 levels in peripheral B cells. (C) miR-98 levels in B cells after exposure to cortisol in the culture for 48 h (the data B were summarized from 3 independent experiments). Data of bars are presented as mean ± SD. *p < 0.01, compared with the healthy subjects (A) or the dose 0 group (C).

Figure 4. Assessment of the effects of miR-98 on suppression of IL-10 expression in B cells

(A, B) the levels of IL-10 mRNA (A) and protein (B) in B cells after treatment in the culture as denoted on the X axis. (C) the bars show the results of miR-98 RNAi of B cells. (D) the bars show the results of miR-92a RNAi of B cells. Data of bars are presented as mean ± SD. *p < 0.01, compared with the saline group. (E, F) representative images show that B cells were transduced with miR-98 shRNA (E) or miR-92a shRNA (F) (the shRNA carried a GFP gene; the green color indicates the GFP protein). (G) an image of naive B cells. The data were summarized from 3 independent experiments. Original magnification of E–G: '630.

Figure 5. Survival allograft hearts

(A–C) the survival curves show the survival rates of mice received allograft heart transplantation. The flow cytometry plots show the frequency of B10 cells in the mouse spleen after heart transplantation. The treatments are denoted above each panel. Anti-miR-98 (anti-miR-92a, or control miR): Mice were also anti-miR-98 liposomes (or anti-miR-92a liposomes, or control liposomes). B10 cells (Naïve cells): Mice received B10 cells (or naïve B cells) at 10⁶ cells/mouse one day before surgery. Cortisol inhibitor (B), or B10 cells (C), or cortisol inhibitor or control materials. Each group consists of 10 mice.

Figure 6. Induction of B10 cells

Naive B cells were isolated from the mouse spleen and cultured for 3 days in the presence of saline, or LPS (1 μg/ml) and anti-CD40 antibody (20 ng/ml). (A) the IL-10 mRNA in the B cells (by RT-qPCR). (B) the IL-10 protein in B cells (by Western blotting). (C) the frequency of B10 cells (by flow cytometry). The data represent 3 independent experiments.