Selenium supplementation suppresses immunological and serological features of lupus in B6.Sle1b mice

Abstract

Systemic lupus erythematosus (SLE) is a debilitating multi-factorial immunological disorder characterized by increased inflammation and development of anti-nuclear autoantibodies. Selenium (Se) is an essential trace element with beneficial anti-cancer and anti-inflammatory immunological functions. In our previous proteomics study, analysis of Se-responsive markers in the circulation of Se-supplemented healthy men showed a significant increase in complement proteins. Additionally, Se supplementation prolonged the life span of lupus prone NZB/NZW-F1 mice. To better understand the protective immunological role of Se in SLE pathogenesis, we have investigated the impact of Se on B cells and macrophages using in vitro Se supplementation assays and the B6.Sle1b mouse model of lupus with an oral Se or placebo supplementation regimen. Analysis of Se-treated B6.Sle1b mice showed reduced splenomegaly and splenic cellularity compared to untreated B6. Sle1b mice. A significant reduction in total B cells and notably germinal center (GC) B cell numbers was observed. However, other cell types including T cells, Tregs, DCs and pDCs were unaffected. Consistent with reduced GC B cells there was a significant reduction in autoantibodies to dsDNA and SmRNP of the IgG2b and IgG2c subclass upon Se supplementation. We found that increased Se availability leads to impaired differentiation and maturation of macrophages from mouse bone marrow derived progenitors in vitro. Additionally, Se treatment during in vitro activation of B cells with anti-CD40L and LPS inhibited optimal B cell activation. Overall our data indicate that Se supplementation inhibits activation, differentiation and maturation of B cells and macrophages. Its specific inhibitory effect on B cell activation and GC B cell differentiation could be explored as a potential therapeutic supplement for SLE patients.

Keywords: B cells; germinal center; macrophages; selenium; systemic lupus erythematosus.

Figure 1.. Selenium supplementation reduces germinal center B cells in B6.Sle1b mice.

B6.Sle1b mice were administered PBS (UT) or MSeA orally starting at 2 mo of age. At 5 mo of age, mice were sacrificed and flow cytometry analyses were performed. (A) Determination of total body weight at indicated time points. (B) Analysis of spleen weight at 5 mo of age. (C) Total number of cells per spleen. (D) Comparative cytometric analysis of total macrophage (CD19^-CD3^-Ly6G^-Ly6C^-SSC^loCD11b⁺F4/80⁺) numbers per spleen (E) Cytometric analysis of total CD19⁺ B220⁺ B cells in the spleen (F) Representative flow plots showing gating strategy of B220⁺ GL-7^hi CD95^hi GC B cells. (G) Total number of B cells in GCs per spleen. Each symbol represents value in a mouse and horizontal lines indicate mean values. (NS, not significant; *, p<0.05).

Figure 2.. Selenium supplementation does not affect monocytes, dendritic cells and granulocytes in B6.Sle1b mice.

B6.Sle1b mice were orally administered PBS (UT) or MSeA starting at 2 mo of age. At 5 mo of age mice were sacrificed and the percentages of cells were analyzed by flow cytometry. (A) CD11b⁺ SSC^loLy6C⁺ Ly6G^- Monocytes. (B) CD11c^hi MHC-II^hi-DCs (C) CD11c^int B220⁺PDCA1⁺-pDCs. (D) CD4⁺ CD25⁺ FOXP3⁺ Tregs. (E) CD11b⁺ SSC^hiLy6C⁺ Ly6G⁺ Neutrophils. (F) CD11b⁺ SSC^hiLy6C⁺ Ly6G^- Eosinophils. (G) CD11b⁺ SSC^loLy6C^hi Ly6G^- naïve monocytes and CD11b⁺ SSC^loLy6C^lo Ly6G^- - inflammatory monocytes in the blood.

Figure 3.. Selenium supplementation reduces the production of autoantibodies in B6.Sle1b mice.

(A) Representative microscopy images of HEp-2 ANA analysis using sera from B6.Sle1b mice, untreated (UT) or at 1 mo, 2 mo and 3 mo after MSeA treatment. (B) Quantification of total ANA fluorescence intensity from 5 mice per indicated group. Each sample was analyzed in duplicate and average values were plotted. (C) Analysis of serum titers of total anti-dsDNA IgG (upper panel) and anti-SmRNP IgG (lower panel) from UT or MSeA-treated B6.Sle1b mice by ELISA. (D-G) Analysis of serum for anti-dsDNA IgG2b (D), anti-dsDNA IgG2c (E), anti-SmRNP IgG2b (F) and anti-SmRNP IgG2c (G) from UT or MSeA treated B6.Sle1b mice by ELISA, at the indicated time after MSeA treatment. Each symbol represents value in a mouse and horizontal lines indicate mean values for panels D-G.

Figure 4.. Selenium suppresses B cell activation in vitro.

Purified WT B cells were left unactivated (UA) or activated with anti-IgM and anti-CD40 for 24h in the absence or presence of indicated MSeA concentrations, after which the cells were analyzed by flow cytometry. gMFI of surface marker: (A) IgD, (B) MHC-II, (C) CD69, (D) CD80 and (E) CD86 expression on B cells. Data are representative of three independent experiments with similar results.

Figure 5.. Effect of various selenium compounds on macrophage differentiation and maturation.

Bone marrow cells were differentiated into macrophages in vitro, in the absence or presence of various Se compounds at the indicated concentrations. After 7 days of differentiation the macrophages were analyzed by flow cytometry. Effect of (A) MSeA, (B) SM, (C) MSC and (D) Sel on the surface expression of CD11b, F4/80, CD86 and MHC-II measured as geometric mean fluorescence intensity (gMFI). Data are representative of two independent experiments with similar results.

Figure 6.. Selenium suppresses REDD1 levels in human monocyte derived macrophages under hypoxic conditions.

(A) Human monocytes were purified by MACS sorting from PBMCs and differentiated into macrophages in vitro. (B) Histograms show surface expression of CD11b, CD86, MHCII, CD68 and MerTK on in vitro differentiated macrophages. (C) In vitro differentiated macrophages were incubated with MSeA under hypoxia (1% oxygen) and REDD1 protein levels were measured by Western blot. β-actin was used as a loading control for protein content.