Impact of dietary vitamin D on immunoregulation and disease pathology in lupus-prone NZB/W F1 mice

Abstract

Vitamin D (VD) deficiency is a highly prevalent worldwide phenomenon and is extensively discussed as a risk factor for the development of systemic lupus erythematosus (SLE) and other immune-mediated diseases. In addition, it is now appreciated that VD possesses multiple immunomodulatory effects. This study aims to explore the impact of dietary VD intake on lupus manifestation and pathology in lupus-prone NZB/W F1 mice and identify the underlying immunological mechanisms modulated by VD. Here, we show that low VD intake accelerates lupus progression, reflected in reduced overall survival and an earlier onset of proteinuria, as well higher concentrations of anti-double-stranded DNA autoantibodies. This unfavorable effect gained statistical significance with additional low maternal VD intake during the prenatal period. Among examined immunological effects, we found that low VD intake consistently hampered the adoption of a regulatory phenotype in lymphocytes, significantly reducing both IL-10-expressing and regulatory CD4⁺ T cells. This goes along with a mildly decreased frequency of IL-10-expressing B cells. We did not observe consistent effects on the phenotype and function of innate immune cells, including cytokine production, costimulatory molecule expression, and phagocytic capacity. Hence, our study reveals that low VD intake promotes lupus pathology, likely via the deviation of adaptive immunity, and suggests that the correction of VD deficiency might not only exert beneficial functions by preventing osteoporosis but also serve as an important module in prophylaxis and as an add-on in the treatment of lupus and possibly other immune-mediated diseases. Further research is required to determine the most appropriate dosage, as too-high VD serum levels may also induce adverse effects, possibly also on lupus pathology.

Keywords: SLE; autoimmune; autoimmunity; choleacliferol; diet; lupus; mice; vitamin D.

Figure 1

Low vitamin D (VD) intake, starting at 5 weeks of age, mildly accelerates disease progression in female lupus-prone NZB/W F1 mice. (A) Serum 25(OH)D₃ concentrations were determined in 19–20-week-old female mice (n = 15–17), fed either a low-, normal-, or high-VD diet, with the latter containing 76,500 IU/kg. (B) Overall survival (OS) of LVD mice (n = 17), NVD mice (n = 18) and HVD mice (n = 18). The Kaplan–Meier method was used for estimating OS. Disease progression was monitored, including (C) proteinuria development, which was assessed semiquantitatively once a week, as well as serum (D) anti-dsDNA IgG and (E) total IgG titers, which were determined at an age of 19–20 weeks and 28–30 weeks in LVD mice (n = 13–15), NVD mice (n = 13–18), and (HVD mice (n = 13–18). Results are displayed as (B) survival curves, (C) time curves, and (D, E) scatter plots, with each data point representing an individual mouse in the latter. Data are expressed as (C) percents ± SEM or (D, E) mean ± SEM. P ≤ 0.05 was considered significant, p ≥ 0.2 is indicated as ns, not significant. O.D., optical density; A.U., arbitrary unit; LVD, low vitamin D; NVD, normal vitamin D; HVD, high vitamin D. ** = p < 0.01; **** = p < 0.0001

Figure 2

Low VD intake from the prenatal period onward accelerates disease progression in female lupus-prone NZB/W F1 mice. (A) OS of LVD mice (n = 11) and HVD mice (n = 13). The Kaplan–Meier method was used for estimating OS. Disease progression was monitored, including (B) proteinuria development, which was assessed semiquantitatively once a week, as well as serum (C) anti-dsDNA IgG and (D) total IgG titers, which were determined at an age of 20 and 28 weeks in LVD mice (n = 10–12), and HVD mice (n = 10–13). Results are displayed as (A) survival curves, (B) time curves, and (C, D) scatter plots, with each data point representing an individual mouse in the latter. Data are expressed as (B) percents ± SE or (C, D) mean ± SEM. P ≤ 0.05 was considered significant, p ≥ 0.2 is indicated as ns, not significant. O.D., optical density; A.U., arbitrary unit; LVD, low vitamin D; HVD, high vitamin D. * = p < 0.05

Figure 3

Low VD intake consistently hampers the adoption of a regulatory phenotype in lymphocytes. (A, B) Hematology analysis of granulocytes and eosinophils in the peripheral blood of (A) 12–15-week-old male and (B) 25–26-week-old female NZB/W F1 LVD and HVD mice. (C, D) Frequencies of Tregs, IL-10⁺ CD4⁺ T cells, IL-10⁺ B cells, IFN-γ⁺ CD4⁺ T cells, IL-17⁺ CD4⁺ T cells, and cDCs in the spleen of (C) 12–15-week-old LVD and HVD male NZB/W F1 mice, as well as (D) 25–26-week-old LVD and HVD female NZB/W F1 mice. Results are displayed as scatter plots, with each data point representing an individual mouse. Data are expressed as mean ± SEM. N/group: males = 7–20; females = 9–13. P ≤ 0.05 was considered significant, p ≥ 0.2 is indicated as ns, not significant. Tregs, regulatory T cells; cDCs, conventional dendritic cells; LVD, low vitamin D; HVD, high vitamin D. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001.

Figure 4

Low VD intake has no consistent effects on cytokine production in innate immune cells. (A, B) Fold gene expression of pro-inflammatory cytokines, following stimulation with 50 ng/ml LPS for 2 h 45 min, in (A) splenic CD11b⁺ innate immune cells and (B) peritoneal macrophages of 12–15-week-old male NZB/W F1 mice. (C, D) Cytokine levels in culture supernatants of (C) BM-derived innate immune cells (+ GM-CSF) and (D) BMDMs (+ M-CSF) from 12–15-week-old male NZB/W F1 mice, following stimulation with either 50 ng/ml LPS and 50 ng/ml Pam3CSK4, or 1 µM CpG and 1 µM R848, for 20 h, normalized to total protein content of adherent cells. Results are displayed as scatter plots, with each data point representing an individual mouse. Data are expressed as mean ± SEM. N/group: LVD = 9-11; HVD = 6-9. P ≤ 0.05 was considered significant, p ≥ 0.2 is indicated as ns, not significant. BM, bone marrow; BMDMs, BM-derived macrophages; Pam3, Pam3CSK4; unstim., unstimulated; LVD, low vitamin D; HVD, high vitamin D. * = p < 0.05

Figure 5

Low VD intake has no effect on phagocytic capacity and surface expression of phagocytosis-associated markers in macrophages. (A, C) Phagocytic uptake of apoptotic thymocytes by (A) peritoneal macrophages and (C) BM-derived macrophages (BMDMs) of 12–15-week-old LVD (n = 10) and HVD (n = 11-12) male animals. (B, D) Surface expression of phagocytosis-associated markers by (B) peritoneal macrophages and (D) BMDMs of 12–15-week-old LVD (n = 7-10) and HVD (n= 10-12) male animals. Results are displayed as scatter plots, with each data point representing an individual mouse. Data are expressed as mean ± SEM. P ≤ 0.05 was considered significant, p ≥ 0.2 is indicated as ns, not significant. MFI, mean fluorescence intensity; BMDMs, BM-derived macrophages; LVD, low vitamin D; HVD, high vitamin D.