Decreased Dicer expression is linked to increased expression of co-stimulatory molecule CD80 on B cells in multiple sclerosis

Abstract

Background: Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system. B cells have been strongly implicated in disease pathogenesis based on clinical trials with B-cell ablation. There is a growing body of evidence linking microRNAs with regulation of the immune system. Dicer, a key enzyme involved in microRNA biogenesis, is necessary for normal B-cell function.

Objective: We aimed to determine whether Dicer expression is impaired in B cells and is linked to increased expression co-stimulatory molecules in patients with MS.

Methods: B cells were separated from blood samples of MS patients and healthy subjects. Expression of Dicer and co-stimulatory molecules CD80 and CD86 was tested. The effect of Dicer modulation on CD80 and CD86 expression in B cells was studied.

Results: Dicer expression was decreased in B cells but not in monocytes of patients with MS compared with healthy subjects. CD80 and CD86 expression was increased on B cells of MS patients compared with healthy subjects. Inhibition of Dicer expression in B cells by small interfering RNA led to increased expression of CD80.

Conclusion: Dicer expression is decreased and is mechanistically linked to increased expression of co-stimulatory molecule CD80 in B cells of patients with MS. This may contribute to activation of immune responses in MS.

Keywords: B cells; CD80; Dicer; Multiple sclerosis.

Figure 1. Dicer protein expression is decreased in B cells of MS patients

B cells were separated from 12 patients with multiple sclerosis (MS) and 11 healthy donors (HD) and tested for Dicer expression by Western blot as described in Material and Methods. Dicer protein expression was normalized against endogenous beta-actin protein. Figure 1A depicts Western blot from a representative HD and a patient with MS. The mean relative expression of Dicer is shown in Figures 1B. * − p = 0.0108.

Figure 2. Expression of CD80 and CD86 on B cells is increased in MS patients

PBMC cells were separated from patients with multiple sclerosis (MS) and healthy donors (HD). CD80 and CD86 expression was analyzed among CD19 positive (gated) B cells. Fig. 2A and 2B depict overlap histograms of CD80 (Fig 2A) and CD86 (Fig 2B) from a representative healthy donor (black histogram) and a patient with MS (white histogram). The Mean Fluorescent Intensity (MFI) of CD80 and CD86 on B cells separated from six healthy donors and five MS patients is shown in Fig. 2C. * − p< 0.02

Figure 3. CD80 expression is increased upon silencing Dicer

Ramos B cells or B cells from healthy human subjects were transfected with Dicer siRNA or control siRNA. Dicer expression was tested by Western blot. CD80 and CD86 expression was examined by Flow cytometry as described in Material and Methods. Fig. 3A depicts Ramos B cells Western blots from a representative experiment. The average fold change of CD80 and CD86 expression from six different experiments with Ramos B cells is shown in Fig. 3B (* − p = 0.0167). The average fold change of CD80 and CD86 expression from experiments with six different human subjects is shown in Fig. 3C (* − p = 0.027).