A Novel MicroRNA-132-Surtuin-1 Axis Underlies Aberrant B-cell Cytokine Regulation in Patients with Relapsing-Remitting Multiple Sclerosis

<div><p>Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.</p></div

SIRT1 regulates LT and TNFα production from B cells.

<p>A: Levels of lymphotoxin (LT), tumor necrosis factor (TNF)α, and interleukin (IL)-10 produced by B cells from healthy subjects (HS: n = 6) treated with the selective pharmacological inhibitor of sirtuin (SIRT)-1, EX-527 (10 µM), or vehicle (0.1% DMSO), and stimulated through the B-cell antigen receptor and CD40 (Wilcoxon test). B: Levels of LT, TNFα, and IL-10 produced by B cells from MS patients (n = 10) treated with the small molecule activator of SIRT1, resveratrol (10 µM), or vehicle (0.1% DMSO), and stimulated through the B-cell antigen receptor and CD40 (Wilcoxon test). C: Levels of LT and TNFα produced by B cells from MS patients (n = 10) treated with either vehicle control (0.1% DMSO) or resveratrol (10 µM) and those from HS (n = 6) treated with vehicle control are shown. NS: not significant (unpaired t-test).</p

Activated B cells of MS patients express lower levels of SIRT1.

<p>A: Expression levels of SIRT1 mRNA in B cells immediately after isolation (Fresh), or when kept in culture for 48 hours either unstimulated (US), stimulated through CD40 (CD40), or stimulated through the BCR and CD40 (BCR+CD40) (Mann-Whitney U-test). B: Frequency of SIRT1⁺ B cells (CD20⁺) within whole PBMC that were kept unstimulated (US: left panel) or stimulated through the BCR and CD40 (BCR+CD40: right panel) (Mann-Whitney U-test).</p

MS B cells express increased levels of miR-132, in association with an abnormal cytokine profile.

<p>A: Levels of lymphotoxin (LT), tumor necrosis factor (TNF)α, and interleukin (IL)-10 (CD40: following CD40 stimulation alone; BCR+CD40: following dual B-cell antigen receptor and CD40 stimulation) in the culture supernatants of B cells from healthy control subjects (HS, n = 13) and untreated MS patients (n = 14) (unpaired t-test). The proportion of memory cells among total B cells, and expression levels of CD40 were not different between B cells from MS patients and HS (data not shown). B: Expression level of miR-132 in B cells either immediately after isolation (Fresh), or following 48 hours in culture when left unstimulated (US), stimulated through CD40 alone (CD40), or stimulated through both the B-cell antigen receptor and CD40 (BCR+CD40). HS n = 13, MS n = 14; (Mann-Whitney U-test).</p

miR-132 enhances LT and TNFα production in association with SIRT1 suppression in B cells.

<p>A: Levels of lymphotoxin (LT), tumor necrosis factor (TNF)α, and interleukin (IL)-10 in B cells from healthy subjects (HS: n = 7) transfected with miR-132 mimic or negative control (NC), and stimulated through the B-cell antigen receptor (BCR) and CD40 (Wilcoxon test). B: Protein level of sirtuin (SIRT)-1 in B cells from HS transfected with miR-132 mimic or NC. A representative result of 2 experiments is shown. The arrow and the arrowhead indicate the bands corresponding to the molecular weight of SIRT1 and β-actin, respectively. C: Level of SIRT1 mRNA in B cells from HS transfected with miR-132 mimic or NC (n = 5) (Paired t-test).</p