nutrients

Article
Low Levels of Vitamin D Promote Memory B Cells
in Lupus
Erin A. Yamamoto 1,2, * , Jane K. Nguyen 3 , Jessica Liu 2 , Emma Keller 2 , Nicole Campbell 2 ,
Cun-Jin Zhang 2 , Howard R. Smith 4 , Xiaoxia Li 2 and Trine N Jørgensen 2, *
1 Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA
2 Lerner Research Institute, Department of Inflammation and Immunity, Lerner Research Institute, Cleveland
Clinic, Cleveland, OH 44195, USA
3 Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH 44195, USA
4 Department of Rheumatologic and Immunologic Disease, Cleveland Clinic, Cleveland, OH 44195, USA
* Correspondence: yamamote@ohsu.edu (E.A.Y.); jorgent@ccf.org (T.N.J.); Tel.: +1-216-444-7454 (T.N.J.)




Received: 19 December 2019; Accepted: 19 January 2020; Published: 22 January 2020


Abstract: Background: Vitamin D deficiency is a known risk factor for Systemic Lupus Erythematosus
(SLE), yet clinical trials have not demonstrated efficacy and few studies have utilized lupus models
to understand the mechanism underlying this relationship. The Act1-/- mouse is a spontaneous
model of lupus and Sjögren’s syndrome, characterized by increased Th17 cells and peripheral B
cell expansion. Vitamin D3 has anti-inflammatory properties, reduces Th17 cells and impairs B
cell differentiation/activation. Therefore, we assessed how varying amounts of vitamin D3 affected
lupus-like disease in the Act1-/- mouse. Methods: Act1-/- mice were fed either low/restricted (0 IU/kg),
normal (2 IU/kg), or high/supplemented (10 IU/kg) vitamin D3 chow for 9 weeks, after which lupus-like
features were analyzed. Results: While we found no differences in Th17 cells between vitamin D3
groups, vitamin D3 restriction specifically promoted memory B cell development, accompanied
by elevated levels of serum IgM, IgG1, IgG3, and anti-dsDNA IgG. A similar significant negative
association between serum vitamin D and memory B cells was confirmed in a cohort of SLE patients.
Conclusion: Low levels of vitamin D3 are associated with elevated levels of memory B cells in an
animal model of lupus and well-controlled SLE patients.

Keywords: Systemic Lupus Erythematosus; vitamin D3; memory B cells; Th17 cells; Act1

1. Introduction
Systemic Lupus Erythematosus (SLE) is an autoimmune disease predominantly affecting women
of child-bearing age at a rate of 20 to 150 cases per 100,000 population [1]. Genetic, environmental, and
hormonal factors all contribute to SLE pathogenesis, and no single factor elicits disease if encountered
alone. Different combinations of risk factors lead to variable and unique patient presentations, including
photosensitivity, rash, arthritis, cytopenias, serositis, nephritis, fatigue, and psychosis [1]. While
the pathophysiology of SLE is incompletely understood, it is characterized by aberrant T and B cell
activity, elevated autoantibody titers, and subsequent organ damage. The characteristic presence of
autoantibodies (e.g., anti-dsDNA IgG, anti-chromatin IgG, anti-Sm IgG), support a pathologic role
for B cells in SLE. Additionally, autoantibody titers have been shown to increase with disease activity,
and newer immunotherapies (e.g., rituximab, belimumab) that target B cells have demonstrated some
benefit [2,3].
Vitamin D deficiency is a common laboratory finding in many autoimmune diseases including
SLE [4]. Vitamin D deficiency is inversely correlated with lupus disease activity, and mutations
in the vitamin D receptor (VDR) have been identified in SLE populations [5]. A couple of studies

Nutrients 2020, 12, 291; doi:10.3390/nu12020291 www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 291 2 of 17

have investigated the relationship between vitamin D3 supplementation and SLE in animal models,
demonstrating an overall protective effect ([6–8] and reviewed in [9]). Although evidence from these
studies suggests that vitamin D3 plays a protective role in lupus, clinical supplementation studies in SLE
patients have demonstrated inconclusive therapeutic benefits [10–13]. Thus, whether supplementation
or deficiency of vitamin D3 contributes to disease prevention or susceptibility, respectively, and the
mechanism(s) by which such regulation would occur remains unknown.
Immune cells (monocytes, dendritic cells, macrophages, activated lymphocytes) express VDR and
1α-hydroxylase, the key enzyme that produces the biologically active form of vitamin D3 (1,25(OH)2 D3)
from its precursor 25(OH)D3. Vitamin D3 has immune-modulating properties, such as an ability
to inhibit Th1 and Th17 differentiation, while promoting Treg development [14–16]. Additionally,
vitamin D3 has been shown to inhibit B cell proliferation [17–19], and differentiation into memory
B cells and antibody-secreting plasma cells [20–22]. There is also a reduction in immunoglobulin
production [21,22], suggesting a role for vitamin D3 in regulating differentiation and/or activity of
downstream memory B cells or plasma cells. The specific role of vitamin D3 in B cell differentiation
and autoantibody secretion in animal models of SLE, however, has not been determined.
The Act1 (TRAF3IP2 or CIKS) knockout mouse is a model for SLE and Sjogren’s syndrome [23,24].
Act1 is a key adaptor protein in IL-17R signaling, as well as a negative regulator of BAFF-BAFFR
and CD40-CD40L signaling in B cells [23–26]. The Act1-/- phenotype is associated with increased
Th17 cells and Th17-related cytokines (IL-17A, IL-21, IL-22), expanded peripheral B cell populations,
hypergammaglobulinemia and autoantibody production, as well as splenomegaly, lymphadenopathy,
and mild nephritis. Given the known inhibitory activity of vitamin D3 on both Th17 cells and B
cell differentiation and immunoglobulin production, we hypothesized that the absence of dietary
vitamin D3 would promote disease development while vitamin D3 supplementation would suppress
disease development. To test this hypothesis, we assessed whether 9 weeks of vitamin D3 restriction or
supplementation was sufficient to alter the Act1-/- autoimmune phenotype; specifically, the development
of SLE-like characteristics. We found that dietary vitamin D3 restriction was associated with increased
autoantibody and immunoglobulin production, as well as increased percentages of splenic memory B
cells, while vitamin D3 supplementation had no significant effect on autoantibody levels and B cell
differentiation patterns. Further studies in SLE patients confirmed a negative correlation between
levels of memory B cells and vitamin D3, supporting the pathogenicity of vitamin D3 deficiency.

2. Materials and Methods

2.1. Patient Enrollment

SLE patients seen by a rheumatologist (H.S.) between July 2017 and June 2018 at the Cleveland
Clinic Department of Rheumatologic and Immunologic Disease (ages 18–80) were invited to volunteer
for an ongoing Lupus Registry. Patients were eligible for inclusion if ACR criteria were met. There
were no exclusions based on disease activity, flares, or type of therapy. Demographic information,
medical history, and relevant clinical data were collected and managed using REDCap electronic data
capture tools hosted at the Cleveland Clinic [27]. At this visit, patients provided blood samples that
were processed for serum and peripheral blood mononuclear cells (PBMCs) and frozen at −80 ◦ C,
until later processing of all samples, concurrently. Fourteen random patient samples were selected for
PBMC B cell analysis as described below. All samples were obtained after patients provided written
informed consent and after approval of the study by the Cleveland Clinic Institutional Review Board.

2.2. Vitamin D3 Dietary Intervention and Animal Care

Act1-/- mice on the Balb/c background were bred at the University of North Carolina Gnotobiotic
center, and transferred to specific pathogen-free housing at Lerner Research Institute at 5–7 weeks
of age. All mice were born within 3 weeks of each other. Immediately upon arrival at the Lerner
Research Institute, the mice were divided into 3 dietary treatment groups—0 IU/kg (low), 2 IU/kg
Nutrients 2020, 12, 291 3 of 17

(normal), or 10 IU/kg (high) of vitamin D3/kg chow. The three treatment groups were matched for
age and sex to limit potential biases. Mice (n = 15) were maintained on their assigned diet for the
duration of this 9-week study. Mice were bled for serum at 0, 3, 6, and 9 weeks post-transfer. Due to the
immunodeficiency status of Act1-/- mice, cages were changed twice weekly and Vetropolycin gel was
applied as needed throughout the experiment. Mice had access to hydrogel to prevent dehydration if
necessary. All animal procedures were approved by the Cleveland Clinic Institutional Animal Care
and Use Committee.

2.3. Organ Harvest/Preparation

Tissue samples were both frozen in OCT™ and prepared for paraffin embedding by 24 h fixation
in 10% formalin, followed by 80% ethanol. Spleen, submaxillary gland, and cervical lymph nodes
were weighed prior to preservation. Kidneys were cut in half longitudinally prior to preservation.
Paraffin embedding, sectioning (5 µm), and hematoxylin and eosin staining were performed by the
Lerner Research Institute Histology Core. Periodic Acid Schiff (PAS) staining was performed with the
Richard–Allan scientific PAS stain kit (Thermo Scientific, Waltham, MA, USA).
Histology measurements were performed on H&E and PAS-stained paraffin-embedded kidney
sections. To quantify the mean glomerular area, 8–15 glomeruli per mouse were traced and measured
for area using the Keyence BZ-X700 All-in-one microscope, then averaged for each mouse. A renal
pathologist (J.N.), blinded to the treatment groups, scored H&E and PAS-stained kidneys for the
presence of endocapillary hypercellularity, extracapillary proliferation, immune deposits, tubular
atrophy, tubular casts, tubular dilation, and interstitial fibrosis and inflammation. A scale of 0–5 was
used for each feature analyzed (8), in which 0 is absent, 1 is 1–5%, 2 is 6–13%, 3 is 11–20%, 4 is 21–50%,
and 5 is greater than 50% of the glomeruli/tubules/area of interest, summing to a maximum possible
score of 40.

2.4. Immunofluorescence Staining of Kidney Tissue

Half kidneys were immediately frozen in OCT™ and subsequently sectioned at 5 µm. Following
a brief acetone fixation, sections were stained with anti-IgG or anti-IgM-TexasRed (Southern Biotech,
Birmingham, AL, USA) and anti-C’3-FITC antibodies (Immunology Consultants Laboratory, Inc,
Portland, OR, USA). Images were obtained using the Keyence BZ-X700 Series at a fixed fluorescence
intensity across slides, which was selected to minimize background fluorescence. Mean integrated
fluorescence area for IgG- or IgM-TexasRed was calculated per glomerulus and averaged for each
mouse. A minimum of 5 glomeruli was evaluated per mouse.

2.5. Serum Cytokine Flow Cytometry

Serum was obtained after 9 weeks of treatment and assessed for selected cytokines by a Cytokine
Bead Array (BD Bioscience). Briefly, four Flex Sets containing fluorescent beads with pre-conjugated
antibodies to each cytokine of interest (IL-1α, IL-6, IL-10, IL-17A) and a fluorescent detection agent
were combined and incubated with 50 µl of serum according to the manufacturer’s protocol. Beads
with bound cytokines were run on an LSR flow cytometer (BD Biosciences, San Jose, CA, USA) with
the assistance of the Lerner Research Institute Flow Cytometry Core. Data were analyzed using FlowJo
v10 software.

2.6. Enzyme-Linked Immunosorbent Assay (ELISA)

Serum from mice treated for 3–9 weeks was used for all ELISA experiments. Mouse 1,25(OH)2 D3
and 25(OH)D3 levels were measured by ELISA (MyBioSource and Eagle Bioscience, USA, respectively).
Anti-dsDNA IgG and anti-SSB IgG ELISAs were run according to the manufacturer’s guidelines
(Alpha Diagnostic International, San Antonio, TX, USA). Immunoglobulin ELISAs were performed
as described previously [28]. Briefly, microtiter plates were coated with 2.5µg/mL of unlabeled goat
anti-mouse Ig (Southern Biotech) in PBS and blocked with PBS-gelatin. Mouse serum was diluted
Nutrients 2020, 12, 291 4 of 17

in assay buffer (5 mg/mL bovine γ-globulin, 5% gelatin, 0.05% Tween-20 in PBS) as follows: IgG
(1:200,000), IgG1 (1:100,000), IgG2a (1:100,000), IgG2b (1:200,000), IgG3 (1:75,000), IgA (1:50,000), IgE
(1:200,000), IgM (1:50,000). Samples were added to plates and incubated at room temperature for 90
min, then washed with PBS; secondary HRP-conjugated goat anti-mouse antibodies specific for IgG,
IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, IgM (all Southern Biotech) were added, followed by incubation for
1 h. Assays were visualized using the TMB substrate kit (Thermo Scientific, Waltham, MA, USA) and
read on a Victor 3 plate reader (Perkin Elmer, Waltham, MA, USA) at 450 nm.

2.7. Flow Cytometry

At the time of sacrifice (9 weeks of treatment), spleens were isolated. Approximately 50% of
each spleen was mashed and red blood cells lysed in ACK buffer (0.15 M NH4Cl, 0.01 M KHCO3, 0.2
mM EDTA) for 5 min. Cells were washed and resuspended in PBS for surface staining against CD3
(145-2C11, PE-Cy7), CD4 (GK1.5, APC), CD8 (53-6.7, PerCP-Cy5.5), B220 (RA3-6B2, PerCP-Cy5.5),
CD93/AA4.1 (AA4.1, PE), CD38 (90, FITC), GL7 (GL-7, Biotin), Streptavidin (PE), and/or CD138 (281-2,
PE) (all antibodies from eBioscience, Waltham, MA, USA) for 30 min. Samples stained only for
surface markers were fixed in 1% paraformaldehyde before analysis. A subset of splenocytes was
analyzed for cytokine production after subsequent stimulation with PMA (2 µg/mL) and ionomycin (1
µg/mL) for 4 h at 37 ◦ C, 5% CO2 , the last 2 h in the presence of 0.4 µL/100 µL cells GolgistopTM (BD
Bioscience). Intracellular staining was performed with fluorescence-conjugated antibodies against
RORγt, IL-17A, IL-21, and IL-22 (all from eBioscience). All intracellular stains were performed using
the Foxp3 transcription factor staining buffer kit (eBioscience) and run within 15 h of permeabilization
on the BD LSR Fortessa (BD Biosciences, CA, USA).
Human PBMCs were isolated from SLE patients by Ficoll gradient and stored at −80 ◦ C. On the
day of analysis, PBMCs were thawed, washed and resuspended in human Fc Block (BD Bioscience),
followed by surface staining against CD19 (HIB19, FITC), CD27 (0323, APC), CD38 (HIT2, Pe-Cy7),
and IgD (IA6-2, PE) (all antibodies from eBioscience) in PBS for 30 min. PBMCs were fixed in 1%
paraformaldehyde before being run on the BD LSR Fortessa. All data from human and mouse
experiments were analyzed using Flowjo v10 software.

2.8. Real-Time Reverse-Transcriptase PCR

Splenocytes were isolated from whole spleen as described above and immediately frozen
at −80 ◦ C. RNA was isolated using the RNAeasy Plus Micro Kit (Qiagen, Valencia, CA) and
converted into cDNA using the qScript cDNA SuperMix (Quanta BioSciences, Gaithersburg,
MD, USA). PCR was subsequently performed using 100 ng cDNA and the PerfeCTa® SYBR®
Green FastMix® ROX (Quanta BioSciences) in the 7300 Real-Time PCR System (Applied
Biosystems, Foster City, CA, USA). β-actin was used as the internal control for all RT-PCR
experiments. All primers were created by Integrated DNA Technologies (Skokie, IL). PCR
analyses were performed in triplicate, or duplicate for β-actin. Primer sequences were as
follows: Aicda 50 -GCGGACATTTTTGAAATGGTA-30 and 30 -GGGAGTTTCAGAATCCGGTT-50 ;
Rgs13 50 -ATCTACATCCAGCCACAGTCTC-30 and 30 - TCAGGAGTTGTTGGTACATTTCAG-50 ;
Il6ra 50 -ACACACTGGTTCTGAGGGAC-30 and 30 -GTGGACGGTTGGAACACCAT-50 ; Itga4
50 -TGTGCAAATGTACACTCTCTTCCA-30 and 30 -CTCCCTCAAGATGATAAGTTGTTCAA-50 ; Actb
50 -TGGGAATGGGTCAGAAGGAC-30 and 30 -GGGTCTAGTACAAACTCTGG-50 .

2.9. Statistical Analyses

Comparisons between treatment groups were analyzed by Student’s t-test with Welch’s correction
for non-normally distributed samples. Two-way ANOVA was used to test for statistical differences in
vitamin D3 levels over time in study animals. Associations between vitamin D3 and PBMC isolated
cells were analyzed by linear regression, with reported r2 values after checking for linearity and error
of variance. Statistical analyses and graphs were performed in GraphPad Prism (La Jolla, CA, USA)
 Nutrients 2020, 12, 291 5 of 17

Comparisons between treatment groups were analyzed by Student’s t-test with Welch’s
correction for non-normally distributed samples. Two-way ANOVA was used to test for statistical
differences in vitamin D3 levels over time in study animals. Associations between vitamin D3 and
Nutrients
PBMC2020, 12, 291 cells were analyzed by linear regression, with reported r2 values after checking
isolated 5 of 17for

linearity and error of variance. Statistical analyses and graphs were performed in GraphPad Prism
or(La
JMPJolla,
Pro CA, USA)
version 14or JMPInstitute,
(SAS Pro version 14NC,
Cary, (SASUSA).
Institute, Cary, NC,
Significance USA).
level wasSignificance
set at alphalevel was
= 0.05 forset
at alpha
all analyses.= 0.05 for all analyses.

3. 3. Results
Results

3.1. Serum
3.1. 25(OH)D3
Serum Levels
25(OH)D3 Plateau
Levels after
Plateau Controlled
after Dietary
Controlled Vitamin
Dietary D3D3
Vitamin Intake
Intake
Act1-deficient
Act1-deficient mice develop
mice developa lupus-like disease
a lupus-like characterized
disease by hyper-Th17
characterized differentiation
by hyper-Th17 and
differentiation
andhyperplasia.
B cell B cell hyperplasia.
Using Using this model,
this model, we analyzed
we analyzed the immunoregulatory
the immunoregulatory effect
effect of of vitamin
vitamin D3. It
D3. It was
was previously
previously reported reported that serum
that serum 25(OH)D3 25(OH)D3 levels plateau
levels plateau after 5 after
weeks 5 weeks of dietary
of dietary intervention
intervention [29].
[29]. Therefore,
Therefore, to ensure to ensure stabilization
stabilization of vitamin
of vitamin D3 all
D3 levels, levels,
miceall mice underwent
underwent 9 weeks9 ofweeks of treatment
treatment with
with
low low chow),
(0 IU/g (0 IU/gnormal
chow),(2normal (2 IU/g
IU/g chow), chow),
or high (10 or
IU/ghigh (10 vitamin
chow) IU/g chow) vitamin
D3 diets prior D3 diets prior
to analysis. We to
analysis. We
determined levelsdetermined levels of serum
of serum 25(OH)D3 25(OH)D3
as an indicator as an indicator
of vitamin D3 status, of as
vitamin D3 status,active
the hormonally as the
hormonally
1,25(OH) 2 D3 active
is 1,25(OH)
tightly regulated
2 D3 is
withtightly
a regulated
half-life in thewith
ordera half-life
of hours in the order
[30,31]. of
Serum hours [30,31].
25(OH)D3 Serum
levels
25(OH)D3 differed
significantly levels significantly differed between
between treatment treatment
groups already groups
after 3 weeks already after 3 weeks
and remained stableand remained
thereafter
stable1A,B).
(Figure thereafter (Figure 1A,
As anticipated, B). were
there As anticipated,
no differencesthere were no
in serum differences
1,25(OH) in serum
2 D3 between the1,25(OH)
groups of2D3

between
mice (Figure the groups of mice (Figure 1C).
1C).

Figure
Figure1. 1.25(OH)D3
25(OH)D3chowchowalters
altersserum
serum 25(OH)D3,
25(OH)D3, but not 1,25(OH)
1,25(OH)22D3 D3levels.
levels.(A)
(A)Longitudinal
Longitudinal and
and(B)(B) 9-weekassessment
9-week assessmentofofserum
serum 25(OH)D3
25(OH)D3 levels in Act1-/-
levels in Act1-/-mice.
mice.(C)
(C)9-week
9-weekanalysis
analysisofof
serum
serum
1,25(OH)
1,25(OH)2 D32D3levels
levels Act1-/-
inin mice.
Act1-/- mice.InIn
(A), each
(A), eachline represents
line represents a single mouse,
a single mouse,and andp-value reflects
p-value reflects
two-way ANOVA, and in (B,C), each symbol represents an individual mouse
two-way ANOVA, and in (B,C), each symbol represents an individual mouse with a horizontal with a horizontal line
line
representing
representing thethe
group
groupmean. * p*<p 0.05,
mean. < 0.05, p<
** ** p <0.01;
0.01;unpaired
unpairedt-test
t-testwith
withWelch’s
Welch’scorrection.
correction.

3.2. Vitamin D3 Manipulation Does Not Affect Th17 Cells in the Act1-/- Mouse
3.2. Vitamin D3 Manipulation Does Not Affect Th17 Cells in the Act1-/- Mouse
Th17 cells were identified as key contributors to the Act1-/- phenotype [32–34]. Furthermore, it was
Th17 cells were identified as key contributors to the Act1-/- phenotype [32–34]. Furthermore, it
shown that inhibition of Th17-related cytokines (IL-17A, IL-21, IL-22) prevented specific autoimmune
was shown that inhibition of Th17-related cytokines (IL-17A, IL-21, IL-22) prevented specific
features [32,33]. Since vitamin D3 has been shown to reduce Th17 differentiation in vitro and
autoimmune features [32,33]. Since vitamin D3 has been shown to reduce Th17 differentiation in vitro
in vivo [14,35–37], vitamin D3 was proposed as a potential therapy for the Act1-/- mouse. Contrary to
and in vivo [14,35–37], vitamin D3 was proposed as a potential therapy for the Act1-/- mouse.
previous studies, dietary differences in vitamin D3 did not alter the Th17 cell population (CD4+RORγt+)
Contrary to previous studies, dietary differences in vitamin D3 did not alter the Th17 cell population
in the Act1-/- mouse model (Figure 2A; see Supplemental Figure S1 for gating strategy). Additionally,
PMA/ionomycin-stimulated Act1-/- splenocytes demonstrated comparable IL-17A production between
vitamin D3 groups (Figure 2B), which corresponded with no difference in serum IL-17A between
treatment groups (Figure 2C). This was further supported by a lack of difference in serum IL-6 (critical
 Nutrients 2020, 12, 291 6 of 17

(CD4+RORγt+) in the Act1-/- mouse model (Figure 2A; see supplemental figure 1 for gating strategy).
Additionally, PMA/ionomycin-stimulated Act1-/- splenocytes demonstrated comparable IL-17A
production between vitamin D3 groups (Figure 2B), which corresponded with no difference in6serum
Nutrients 2020, 12, 291 of 17
IL-17A between treatment groups (Figure 2C). This was further supported by a lack of difference in
serum IL-6 (critical for Th17 differentiation) levels between treatment groups (Figure 2C). It should
for
beTh17
noteddifferentiation)
that despite levels
a lackbetween treatment
of association withgroups (Figure
vitamin 2C). It should
D3 levels, be noted
a positive that despite
correlation betweena
lack of association with vitamin D3 levels, a positive correlation between numbers of circulating
numbers of circulating Th17 cells and serum levels of IL-6 and IL-17A was observed as expected Th17
cells and serum
(Figure 2D,E). levels of IL-6 and IL-17A was observed as expected (Figure 2D,E).

Figure
Figure 2. 2.Dietary
Dietaryvitamin
vitaminD3 D3manipulation
manipulation does
doesnot
notaffect Th17
affect Th17cells inin
cells the
theAct1-/-
Act1-/-mouse.
mouse.(A) Splenic
(A) Splenic
Th17 cells (CD3+CD4+RORγt+) and (B) IL-17A production by splenic Th17
Th17 cells (CD3+CD4+RORγt+) and (B) IL-17A production by splenic Th17 cells stimulated with cells stimulated with PMA
PMA
and ionomycin. (C) IL-1α, IL-6, and IL-17A cytokine profile after 9 weeks of dietary
and ionomycin. (C) IL-1α, IL-6, and IL-17A cytokine profile after 9 weeks of dietary vitamin D3. (D,E) vitamin D3. (D,E)
Association
Association between
between splenic
splenic circulating
circulating Th17
Th17 cells and
cells andserum
serum levels
levelsofof
IL-6
IL-6(D)
(D)and IL-17A
and IL-17A(E). Each
(E). Each
symbol represents an individual mouse. In (A,B), the mean of each group is
symbol represents an individual mouse. In (A,B), the mean of each group is represented by a represented by a horizontal
line, and in (D,E)
horizontal a best
line, and infit line aisbest
(D,E) shown.
fit line is shown.
3.3. Vitamin D3 Restriction Specifically Augments Memory B Cells
3.3. Vitamin D3 Restriction Specifically Augments Memory B Cells
It was previously shown that the Act1-/- mouse displays increased numbers of peripheral
It was previously shown that the Act1-/- mouse displays increased numbers of peripheral B cells
B cells and plasma cells due to Act10 s role as a negative regulator of B cell survival and
and plasma cells due to Act1′s role as a negative regulator of B cell survival and expansion [23,25,26].
expansion [23,25,26]. Furthermore, it is well-established that Act1-/- mice develop elevated levels
Furthermore, it is well-established that Act1-/- mice develop elevated levels of serum autoantibodies
of serum autoantibodies [23,25,38]. Splenic B cell populations were analyzed to assess the role of
[23,25,38]. Splenic B cell populations were analyzed to assess the role of vitamin D3 in peripheral B
vitamin D3 in peripheral B cell populations and autoantibody production (see Supplemental Figure
cell populations and autoantibody production (see supplemental figure 2 for gating strategy). We
S2 for gating strategy). We found no differences in total B220+ B cells, as well as no differences in
found no differences in total B220+ B cells, as well as no differences in transitional T1
transitional T1 (B220+AA4.1+IgM+CD23-; p = 0.71), T2 (B220+AA4.1+IgM+CD23+; p = 0.38), or T3
(B220+AA4.1+IgM+CD23-; p = 0.71), T2 (B220+AA4.1+IgM+CD23+; p = 0.38), or T3 (B220+AA4.1+IgM-
(B220+AA4.1+IgM-CD23+; p = 0.22) B cell subsets between treatment groups (Figure 3A–D). Germinal
CD23+; p = 0.22) B cell subsets between treatment groups (Figure 3A–D). Germinal center (GC) B cells
center (GC) B cells (B220+IgM-GL7+CD38low) were also similar across vitamin D3 groups (low vs.
(B220+IgM-GL7+CD38low) were also similar across vitamin D3 groups (low vs. normal, p = 0.66; low
normal, p = 0.66; low vs. high, p = 0.38; normal vs. high, p = 0.34) (Figure 3E,F); however, the vitamin
vs. high, p = 0.38; normal vs. high, p = 0.34) (Figure 3E,F); however, the vitamin D3-restricted group
D3-restricted group displayed significantly higher levels of memory B cells (B220+IgM-CD38hiGL7-)
displayed significantly higher levels of memory B cells (B220+IgM-CD38hiGL7-) compared to the
compared to the high vitamin D3 group (p < 0.05) (Figure 3C). Surprisingly, the percentage of
high vitamin D3 group (p < 0.05) (Figure 3C). Surprisingly, the percentage of plasmablasts (B220-
plasmablasts (B220-CD138+IgM+) remained unaffected by vitamin D3 manipulation (low vs. normal,
CD138+IgM+) remained unaffected by vitamin D3 manipulation (low vs. normal, p = 0.46; low vs.
p = 0.46; low vs. high, p = 0.16; normal vs. high, p = 0.21) as did plasma cells (B220-CD138+IgM-; low
high, p = 0.16; normal vs. high, p = 0.21) as did plasma cells (B220-CD138+IgM-; low vs. normal, p =
vs. normal, p = 0.19; low vs. high, p = 0.97; normal vs. high, p = 0.47) (Figure 3G,H). Thus, vitamin D3
0.19; low vs. high, p = 0.97; normal vs. high, p = 0.47) (Figure 3G,H). Thus, vitamin D3 treatment
treatment selectively impaired memory B cells in Act1-/- mice.
selectively impaired memory B cells in Act1-/- mice.
Nutrients 2020, 12, 291 7 of 17
Nutrients 2020, 12, 291 7 of 17

Figure 3. Memory B cells are specifically affected by vitamin D3 restriction. Splenocytes

Figure
were 3. Memory
analyzed B cells are of
for populations specifically
(D) totalaffected
B220+ by B vitamin D3 restriction.
cells, (A–C) transitional Splenocytes
B cells—T1 were
(B220+AA4.1+IgM+CD23-),
analyzed for populations T2 of
B220+AA4.1+IgM+CD23+),
(D) total B220+ B cells, and T3 (B220+AA4.1+IgM-CD23+),
(A–C) transitional B cells—T1
(E)(B220+AA4.1+IgM+CD23-),
germinal center B cells (B220+IgM-GL7+CD38 low ), (F) memory B cells (B220+IgM-CD38hi GL7-),
T2 B220+AA4.1+IgM+CD23+), and T3 (B220+AA4.1+IgM-CD23+), (E)
(G)germinal
plasmablasts (B220-CD138+IgM+)
center and (H) plasma
B cells (B220+IgM-GL7+CD38 low ), cells (B220-CD138+IgM-).
(F) memory (I) Serum IL-10
B cells (B220+IgM-CD38 levels.(G)
hiGL7-),

Each symbol represents

plasmablasts an individual mouse,
(B220-CD138+IgM+) and (H)and the mean
plasma cellsof(B220-CD138+IgM-).
each group is represented by a horizontal
(I) Serum IL-10 levels.
line. * p <symbol
Each p < 0.01; unpaired
0.05, ** represents t-test with
an individual Welch’s
mouse, andcorrection.
the mean GC of = germinal
each groupcenter.
is represented by a
horizontal line. * p < 0.05, ** p < 0.01; unpaired t-test with Welch’s correction. GC = germinal center.
In a gene array study of naïve, germinal center, memory, and plasma cells, Rgs13 expression was
identified
In aasgene
specific
arraytostudy
germinal center
of naïve, B cells, center,
germinal whereas Itga4 and
memory, andIl6ra genecells,
plasma expression was specific
Rgs13 expression was
foridentified
memory B ascells [39].toItgerminal
specific should be noted
center B though that Il6ra
cells, whereas Itga4transcripts
and Il6ra are
genealso widely present
expression in
was specific
T cells and myeloid
for memory B cellscells
[39].[40,41],
It shouldand notedAicda
bewhile though genethatexpression is commonly
Il6ra transcripts are alsoused
widelyas present
a marker in T
forcells
the germinal center B cell as AID (activation-induced deaminase) is critical
and myeloid cells [40,41], and while Aicda gene expression is commonly used as a marker for for class-switching
andthesomatic
germinal hypermutation,
center B cell as Aicda
AIDis(activation-induced
also expressed in memory deaminase) B cells, albeit for
is critical at aclass-switching
lower level [39]. and
Finally, Bcl6 is expressed highly by memory B cells, but also in T follicular helper
somatic hypermutation, Aicda is also expressed in memory B cells, albeit at a lower level [39]. Finally,cells and memory T
cells [42,43].
Bcl6 We found
is expressed no difference
highly by memory in Rgs13 gene
B cells, butexpression levels from
also in T follicular whole
helper spleen
cells and from
memoryvitamin
T cells
D3-manipulated Act1-/-
[42,43]. We found no difference in Rgs13 gene expression levels from whole spleen from vitaminInD3-
mice, supporting a lack of difference in levels of GC B cells (Table 1).
contrast, Aicda and
manipulated Bcl6
Act1-/- genesupporting
mice, expressiona lack
was ofsignificantly
difference in reduced
levels ofinGC theBhigh
cells vitamin
(Table 1).D3In group
contrast,
(Table
Aicda1),and
potentially
Bcl6 gene as expression
a result of the
wasreduced levelsreduced
significantly of memory in theB cells
high(see Figure
vitamin D33C). Itga4
group gene 1),
(Table
expression levels were similarly reduced (p = 0.054) in the high vitamin D3 group,
potentially as a result of the reduced levels of memory B cells (see Figure 3C). Itga4 gene expression further supporting a
reduction in memory
levels were similarlyBreduced
cells. We(pfound noindifferences
= 0.054) in Il6raD3
the high vitamin gene expression
group, in these total
further supporting spleen
a reduction
samples.
in memoryTakenB together,
cells. Wegenefound transcript levels support
no differences a specific
in Il6ra gene effect ofinvitamin
expression D3 on
these total memory
spleen B
samples.
cellTaken
differentiation in Act1-/- mice.
together, gene transcript levels support a specific effect of vitamin D3 on memory B cell
differentiation in Act1-/- mice.
IL-10 is a pleiotropic cytokine with both immunostimulatory and immunoregulatory roles [44].
IL-10 has been found to be involved in the germinal center reaction and as a driver of differentiation
of naïve B cells into memory B cells and plasma cells [45]. We analyzed vitamin D3-treated Act1-/-
Nutrients 2020, 12, 291 8 of 17

Table 1. Whole spleen gene expression.

Vitamin D3
Gene Low Normal High
Rgs13 1.2 ± 0.1 1.1 ± 0.1 1.4 ± 0.2
Bcl6 0.8 ± 0.1 1.0 ± 0.1 0.3 ± 0.02 ac
Aicda 2.5 ± 0.5 2.4 ± 0.9 1.1 ± 0.3 a
Itga4 0.8 ± 0.1 1.0 ± 0.1 0.6 ± 0.2 b
Il6ra 1.3 ± 0.3 1.0 ± 0.1 1.4 ± 0.2
Irf4 1.2 ± 0.1 1.0 ± 0.1 0.8 ± 0.2
a p < 0.05 versus low; b p ≤ 0.05, c p < 0.01 versus normal.

IL-10 is a pleiotropic cytokine with both immunostimulatory and immunoregulatory roles [44].
IL-10 has been found to be involved in the germinal center reaction and as a driver of differentiation
of naïve B cells into memory B cells and plasma cells [45]. We analyzed vitamin D3-treated Act1-/-
mice for serum IL-10 levels and found significantly increased levels in the low and normal vitamin D3
groups as compared with the high group (p < 0.05–0.01) (Figure 3I).

3.4. Elevated Immunoglobulin Production and Anti-dsDNA in Vitamin D3-Restricted Mice

The Act1-/- mouse displays hypergammaglobulinemia, as well as elevations in specific
immunoglobulin subtypes including IgG1, IgG2a, IgG2b, and IgE [23]. After 9 weeks of vitamin D3
manipulation, serum IgM levels were significantly greater in the high vitamin D3 group compared
to both the low and normal vitamin D3 groups (Figure 4A). Oppositely, total IgG levels were found
to be significantly higher in the vitamin D3 low group (Figure 4B), supporting B cell activation in
vitamin D3-restricted Act1-/- mice. We found no statistical differences in serum IgA, IgE, IgG1, or IgG2b
between the groups, although a trend for higher Ig levels in the low vitamin D3 group was identified
for IgA, IgG1 and IgG2b (Figure 4C–E and data not shown). Interestingly, there were significantly
elevated IgG3 levels in the vitamin D3 low group only (Figure 4F), suggesting a specific effect of
vitamin D3 on limiting Ig class switching to IgG3.
Anti-nuclear autoantibodies are classic features of mouse lupus-like disease, and Act1-/- mice at
3.5 months were previously shown to express elevated anti-dsDNA IgG autoantibodies and to have
detectable, albeit very low levels of anti-SSB IgG autoantibodies [24]. We assessed Act1-/- serum for the
presence of anti-dsDNA, anti-SSB and anti-histones IgG autoantibodies. Vitamin D3-restricted mice
displayed significantly elevated serum levels of anti-dsDNA IgG autoantibodies (Figure 4G), while no
differences were detected in anti-SSB and anti-histones IgG levels between the groups (Figure 4H,I).
Despite a lack of significance, it should be noted that 6/7 mice in the low vitamin D3 group displayed
detectable levels of anti-SSB, compared to only 1/4 and 2/4 mice in the normal and high vitamin D3
groups, respectively.
 Nutrients 2020, 12, 291 9 of 17

levels2020,
Nutrients of naïve
12, 291 B cells (CD19+IgD+) (p = 0.16) or plasmablasts (CD19+IgD-CD27+CD38+) (p 9= of0.091)
17
(Figure 6B,D).

Figure
Figure 4. 4. Immunoglobulin
Immunoglobulin and
and autoantibodyproduction
autoantibody productionareareaffected
affectedby byvitamin
vitaminD3 D3 manipulation
manipulation in
Act1-/- mice. (A–F)
in Act1-/- (A–F)Serum
Serumimmunoglobulins
immunoglobulinsand and(G–I)
(G–I)serum
serum autoantibodies
autoantibodies (anti-dsDNA
(anti-dsDNA IgG, anti-
IgG,
SSB IgG,
anti-SSB IgG,and
andanti-histone
anti-histone IgG)
IgG) were measured
measured by
byELISA.
ELISA.Each
Eachsymbol
symbol represents
represents oneone individual
individual
mouse,
mouse, andand
thethe mean
mean of each
of each group
group is represented
is represented by aby a horizontal
horizontal *p<
line.line. * p0.05, ** p**<p0.01;
< 0.05, < 0.01; unpaired
unpaired
t-test
t-test withwith Welch’s
Welch’s correction.
correction.

3.5. Vitamin D3 Exposure Does Not Affect Features of Nephritis after 9 Weeks
Glomerulonephritis in the Act1-/- mouse is typically mild, characterized by IgG and IgM glomerular
deposition at 8–12 months [24]. Analysis of Ig deposition in the kidneys showed similar levels of
IgM-immune complexes in the glomeruli between the treatment groups (Figure 5A,B). We observed
increased IgG deposition in the low vitamin D3 group compared to the high vitamin D3 group, which
corresponded with total IgG/IgG3 and anti-dsDNA IgG patterns in Act1-/- sera (p = 0.05) (Figure 5C).
Quantification of renal histological findings from H&E and PAS-stained kidneys showed no difference
between groups (Figure 5D–F).
Nutrients 2020, 12, 291 10 of 17
Nutrients 2020, 12, 291 10 of 17

Figure 5. Renal immunoglobulin deposition and inflammation is unaffected by vitamin D3 manipulation

in Act1-/- mice. (A) Act1-/- kidneys were stained for IgM (red) and C’3 (green) or IgG (red) and C’3
Figure 5. Renal immunoglobulin deposition and inflammation is unaffected by vitamin D3
(green), and analyzed for (B) IgM and (C) IgG deposition in the glomeruli. Kidneys were also stained
manipulation in Act1-/- mice. (A) Act1-/- kidneys were stained for IgM (red) and C’3 (green) or IgG
by (D) H&E and (E) PAS to assess glomerular and tubular inflammation and injury which is combined
(red) and C’3 (green), and analyzed.
into a renal score (F). Each symbol represents an individual mouse and the mean of each group is
represented by a horizontal line. * p < 0.05, ** p < 0.01; unpaired t-test with Welch’s correction.

3.6. Memory B Cells Are Negatively Associated with Vitamin D3 in SLE

To determine if the specific difference in memory B cell levels observed in Act1-/- mice was also
detectable in human SLE patients, we tested PBMC samples from 14 SLE patients (Table 2). All patients
were well-controlled, with SLEDAI-2K clinical severity scores ranging from 0 to 8. Vitamin D3 levels
ranged from 9.9 to 54.7 ng/mL, with 46% of patients presenting as vitamin D3 deficient (<30 ng/mL as
per institutional laboratory standards). Corresponding with data from the Act1-/- mouse, SLE patient
serum 25(OH)D3 was negatively correlated with memory B cells (CD19+IgD-CD27+CD38-) (p < 0.01)
(Figure 6A,C), while there was no correlation between serum 25(OH)D3 and levels of naïve B cells
(CD19+IgD+) (p = 0.16) or plasmablasts (CD19+IgD-CD27+CD38+) (p = 0.091) (Figure 6B,D).
Nutrients 2020, 12, 291 11 of 17

Table 2. Systemic Lupus Erythematosus (SLE) patient characteristics.

25(OH)D3 SLEDAI anti-dsDNA IL- Th17 %Naive %Memory

Pt Sex Age Race C3/C4 Cr %Plasma-blasts Medication
(ng/mL) -2K (IU/mL) 17A cells B cells B cells
Hydroxychloroquine,
1 F 33 Other 45.1 6 - 114/46 - 14.0 0.4 9.73% 0.24% 0.02%
Mycophenolate
2 F 36 White 38.0 6 n.d. - - 6.0 0.68 9.58% 1.06% 0.03% -
Hydroxychloroquine,
3 F 68 Black 46.1 2 102 - 0.81 10.0 0.91 3.27% 0.49% 0.07%
Oral steroids
Methotrexate, Oral
4 F 57 White 36.4 4 n.d. 160/37 0.62 9.5 0.81 22.44% 0.67% 0.06%
steroids
5 M 70 White 54.7 2 n.d. - 2.07 7.0 0.89 22.65% 0.63% 0.05% Hydroxychloroquine
6 F 54 White 38.9 2 n.d. 146/26 0.77 8.0 0.66 4.24% 0.79% 0.05% Hydroxychloroquine
Hydroxychloroquine,
7 F 36 Other 28.7 6 46 105/14 0.67 64.6 1.42 13.83% 0.94% 0.15%
Oral steroids
Hydroxychloroquine,
8 F 39 Black 13.1 6 n.d. 105/18 0.75 32.7 0.8 8.75% 1.36% 0.05% Mycophenolate, Oral
steroids
Hydroxychloroquine,
9 F 45 White 18.1 4 n.d. 186/35 0.67 1.8 2.19 3.72% 0.74% 0.06%
Oral steroids
Hydroxychloroquine,
10 F 45 White 36.8 4 n.d. 145/44 0.89 23.8 0.76 2.04% 0.81% 0.03%
Oral steroids
11 F 28 Black 9.9 2 n.d. 117/23 0.52 2.2 1 8.52% 1.21% 0.11% -
Hydroxychloroquine,
12 F 39 White 26.8 0 n.d. 145/30 2.63 16.5 1.47 3.10% 0.73% 0.02%
Oral steroids
Hydroxychloroquine,
13 F 39 Other 21.1 3 - 65/12 0.7 17.8 0.56 9.87% 1.60% 0.08%
Mycophenolate
14 F 61 White 36.6 8 12 121/28 0.83 306.2 0.12 7.41% 0.88% 0.04% Oral steroids
Th17 cells (CD4+RORγt+) are reported as percentage of CD4+ cells. Naïve B cells, memory B cells and plasmablasts from peripheral blood mononuclear cells (PBMCs) are reported as
percentage of live cells. Pt = patient; SLEDAI-2K = Systemic Lupus Erythematosus Disease Activity Index-2000; Cr = creatinine; n.d. = not detectable; - = not done.
Nutrients 2020,
Nutrients 12,12,
2020, 291291 12 12
of of
17 17

Figure
Figure 6. 6. Memory
Memory B cells
B cells areare negatively
negatively correlated
correlated with
with vitamin
vitamin D levels
D levels in in
SLESLE patients.
patients. (A)(A) Gating
Gating
strategy
strategy forfor Figure
Figure 25. (OH)D
25(OH)D levels
levels against
against circulating
circulating (B) (B) naïve
naïve B cells
B cells (CD19+IgD+),
(CD19+IgD+), (C)(C) memory
memory BB
cells
cells (CD19+IgD-CD27+CD38-)
(CD19+IgD-CD27+CD38-) andand (D)
(D) plasmablasts
plasmablasts (CD19+IgD-CD27+CD38+)
(CD19+IgD-CD27+CD38+) displayed
displayed asas percent
percent
of of live
live cells.
cells. Each
Each symbol
symbol represents
represents anan individual
individual patient,
patient, n =n 14.
= 14.
PBPB
= =plasmablast.
plasmablast.

4. 4.
Discussion
Discussion
Persistent
Persistent ininthe
theautoimmune
autoimmuneliterature
literatureisisthe
thelack
lackofof clarity
clarity regarding
regarding the role of
the role of vitamin
vitamin D3D3in
inautoimmune
autoimmunedisease. disease.Despite
Despitepervasive
pervasivevitamin
vitaminDDdeficiency
deficiencyacross
acrossautoimmune
autoimmune diseases, there
diseases, there has
has been
been little
little consensus
consensus on aon a mechanism
mechanism explaining
explaining how how
vitaminvitamin D deficiency
D deficiency relatesrelates to disease
to disease features,
features,
and fewand few studies
studies have explored
have explored these mechanisms
these mechanisms in animal in models
animal models of SLE [6–9,46].
of SLE [6–9,46]. In thiswe
In this study,
study, we explored the relationship between low, normal and high vitamin D3
explored the relationship between low, normal and high vitamin D3 exposure and lupus-like disease exposure and lupus-like
disease
in the in the Act1-/-
Act1-/- mousemouse
and inand in a cohort
a cohort of well-controlled
of well-controlled SLE patients.
SLE patients. Interestingly,
Interestingly, we foundwe that
foundlow
that low vitamin D3 levels specifically affected memory B cells and, at least
vitamin D3 levels specifically affected memory B cells and, at least in the mouse model, antibody in the mouse model,
antibody
production.production.
Spontaneous
Spontaneous autoimmune
autoimmune models oftenoften
models require time totime
require develop features features
to develop of autoimmunity. Despite
of autoimmunity.
a short experimental timeframe of 9 weeks, we detected significant elevations
Despite a short experimental timeframe of 9 weeks, we detected significant elevations in anti-dsDNA in anti-dsDNA IgG
antibody levels and
IgG antibody total
levels andimmunoglobulin levels (total
total immunoglobulin IgG(total
levels and IgG3)
IgG andin mice
IgG3)fedina mice
vitaminfedD3-restricted
a vitamin D3-
diet compared
restricted diettocompared
mice fed diets containing
to mice fed dietsnormal or high
containing vitamin
normal D3. Levels
or high vitamin ofD3.
IgA,Levels
IgG1 and IgG2b
of IgA, IgG1
did not reach statistical significance, but nevertheless trended in a similar fashion,
and IgG2b did not reach statistical significance, but nevertheless trended in a similar fashion, while while serum IgM
levels
serum showed a reciprocal
IgM levels showedpattern. Interestingly,
a reciprocal pattern. we observed awe
Interestingly, significant
observeddecrease in serum
a significant IL-10in
decrease
serum IL-10 in the high vitamin D3 group, and IL-10 has been shown to promote IgG1 and IgG3
production by human B cells [47]. Higher levels of IL-10 in the low and normal vitamin D3 groups
Nutrients 2020, 12, 291 13 of 17

in the high vitamin D3 group, and IL-10 has been shown to promote IgG1 and IgG3 production by
human B cells [47]. Higher levels of IL-10 in the low and normal vitamin D3 groups might therefore
contribute to the observed elevations in IgG1 and IgG3. If so, it is interesting that vitamin D3 treatment
in this mouse model led to a reduction in IL-10, thus suggesting that IL-10 plays a pathogenic rather
than anti-inflammatory role in the Act1-/- mouse. A similar relationship has been reported in both
mouse models of lupus and SLE patients [48–52]. Additionally, the low number of mice in each of the
treatment groups must be considered when interpreting these data.
Anti-dsDNA IgG is a strong marker of lupus-like disease and was highly elevated in Act1-/- mice
fed a low vitamin D3 diet. However, no differences were observed between the normal and high
vitamin D3 groups. This finding suggests that a lack of vitamin D3 has a greater impact on disease
than high vitamin D3 supplementation. In fact, the majority of our data suggest that high amounts of
vitamin D3 fail to provide additional benefit compared to an adequate amount of vitamin D3. This
observation is somewhat contradictory to the available data from vitamin D3-manipulated MRL-lpr/lpr
lupus-prone mice [6–8]. One explanation may be that these studies tested the efficacy of active vitamin
D3 analogs (i.e., 1,25(OH)2 D3, 1,24R(OH)2 D3, 22-oxa-1,25(OH)2 D3), as an intervention, while our
study was aimed at evaluating the effect of 25(OH)D3, as this is the form frequently used in clinical
studies [53]. Although vitamin D3 levels were significantly different and stable already after 3 weeks of
treatment, providing the animals with steady-state levels of vitamin D3 for six weeks, it is also possible
that constitutive high vitamin D3 levels are required beyond the limited disease course of 9 weeks.
As such, a longer interventional period might have also elicited measurable differences between the
normal and high vitamin D3 groups. The fact that the Act1-/- mice studied here did not display the
overt splenomegaly or lymphadenopathy commonly seen in Act1-/- mice [23,24] also supports this
explanation (data not shown).
The Act1-/- mouse has an intrinsic drive for Th17 development due to unregulated STAT3 activity
leading to Th17 differentiation [34]. Despite support in the literature for a role for vitamin D3 in
reducing Th17 cell differentiation [14,54,55], we did not observe any changes in the splenic Th17 cell
population, serum IL-17A, or stimulated production of IL-17A. It is possible that the mechanism of
vitamin D3-mediated inhibition of Th17 differentiation occurs upstream of STAT3, which might explain
why no differences in Th17 cells were observed via staining for RORγt. Alternatively, the intrinsic
drive towards Th17 development might have a greater influence on Th17 differentiation than vitamin
D3. Further studies are needed to determine the exact target of vitamin D3 in Th17 differentiation
and function.
Given the correlation between autoantibody titer and SLE disease activity, it is reasonable to
infer that vitamin D30 s association with SLE may be attributable to its role in the regulation of splenic
B cell differentiation. In fact, we found the B cell compartment to be the primary target of vitamin
D3 restriction in the Act1-/- mouse. Vitamin D3 restriction specifically promoted memory B cells,
without affecting germinal center B cells or plasmablasts/plasma cells. Increased Aicda, Bcl6, and
Itga4 expression in the low vitamin D3 group further supported these data. Consistent with these
data, vitamin D3 supplementation demonstrated transcriptional regulation of VDR in the germinal
center and inhibition of Aicda expression (encoding AID, activation-induced deaminase), a cytidine
deaminase critical for class-switch recombination and somatic hypermutation [56]. While expression of
Bcl6 is not limited to B cells, it is interesting to note that the protein is also found in T follicular helper
cells and memory T cells [42,43], and thus reduced expression corresponds with reduced humoral
immune activation in general. The relationship between memory B cells and vitamin D3 was further
supported by a negative correlation between serum 25(OH)D3 and memory B cells, but not total B
cells and plasmablasts, in PBMC samples from SLE patients. Overall, these observations are consistent
with early studies on B cell development that showed inhibition of B cell activation and differentiation
by vitamin D3 metabolites and analogs [17,20–22].
It is puzzling that the observed difference in memory B cells is not mimicked in the populations
of plasmablasts and plasma cells, as would be expected if the memory B cells were a result of T
Nutrients 2020, 12, 291 14 of 17

cell-dependent germinal center activity. T cell-independent memory B cells (B1b cells) have previously
been identified as a population of B cells primarily located in the peritoneal and pleural cavities with
the capacity to develop memory phenotypes and produce immunoglobulins without T cell interactions,
thereby bypassing the germinal center [57]. Therefore, the isolated effect observed on memory B cells
could be a consequence of the development of T cell-independent memory B cells. This possibility
is further supported by the high level of IgG3 identified in the low vitamin D3 group, as it has been
shown that in particular IgG3 is produced in response to T cell-independent type 2 antigens [58,59].
Unfortunately, there are surprisingly few data in the clinical literature regarding memory B cells
and vitamin D3 levels in SLE patients, though evidence from multiple sclerosis (MS) studies has
shown an association between lower intrathecal 25(OH)D3 and increased class-switched memory B
cell accumulation in the CSF during MS relapses [19].
Finally, the origin of vitamin D deficiency in SLE is unknown. It is possible that the vitamin
D deficiency observed in SLE patients is a result of reduced sun exposure due to photosensitivity;
however, such a correlation remains speculative. Regardless of the cause, vitamin D deficiency is
widely believed to play a role in pathogenesis. In support thereof, the few animal studies available
have demonstrated improvement in lupus features when supplemented with 1,25(OH)2 D3 [6,7,60], yet
no improvement was observed when animals were supplemented with 25(OH)D3 [46]. Based on these
studies, it is possible that calcitriol supplementation is needed to achieve clinical improvement. Such a
requirement may explain the lack of clinical success, as calcitriol, but not 25(OH)D3, supplementation
carries a risk of toxicity and is therefore not typically used for SLE patients without severe kidney
disease. Investigations into whether there are therapeutic differences between the two and whether
1,25(OH)2 D3 can be therapeutic at non-toxic concentrations will be interesting.

5. Conclusions
In conclusion, vitamin D3 deficiency has been established as a risk factor for lupus, yet more
studies focus on the effect of supplementation rather than the effect of deficiency. The majority of the
studies on vitamin D3 in mouse models of lupus compare normal vitamin D3 exposure to high levels
of 1,25(OH)2 D3, the hormonally active form of vitamin D3, while clinically, patients are supplemented
with cholecalciferol (inactive vitamin D3), due to the increased risk of hypercalcemia in response to
1,25(OH)2 D3 as compared to cholecalciferol or 25(OH)D3 [61]. Our study is unique in that it compared
the effect of a low vitamin D3 diet with that of both normal and supplemented (high) vitamin D3 states
using 25(OH)D3, the intervention that is also used for treatment clinically. This approach allowed us to
identify memory B cells as a functional result of vitamin D3 restriction in an autoimmune-prone model.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/2/291/s1,

Figure S1: Th17 gating strategy, Figure S2: B cell gating strategy.
Author Contributions: Conceptualization, E.A.Y., X.L., T.N.J.; methodology, E.A.Y., T.N.J.; formal analysis, E.A.Y.;
investigation, E.A.Y., J.K.N., J.L., E.K., N.C.; resources, H.R.S., X.L., T.N.J.; writing—E.A.Y.; writing—review and
editing, H.R.S., X.L., T.N.J.; supervision and administration, H.R.S., C.-J.Z., X.L., T.N.J.; funding acquisition, E.A.Y.,
T.N.J. All authors have read and agreed to the published version of the manuscript.
Funding: E.Y. was funded by the Howard Hughes Medical Institute Medical Research Fellows Program.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Tsokos, G.C. Systemic lupus erythematosus. N. Engl. J. Med. 2007, 369, 587–596.
2. Harvey, P.R.; Gordon, C. B-Cell Targeted Therapies in Systemic Lupus Erythematosus. BioDrugs 2013, 27,
85–95. [CrossRef] [PubMed]
3. Liossis, S.-N.C.; Staveri, C. B Cell-Based Treatments in SLE: Past Experience and Current Directions. Curr.
Rheumatol. Rep. 2017, 19, 78. [CrossRef]
4. Sakthiswary, R.; Raymond, A.A. The Clinical Significance of Vitamin D in Systemic Lupus Erythematosus: A
Systematic Review. PLoS ONE 2013, 8, e55275. [CrossRef]
Nutrients 2020, 12, 291 15 of 17

5. Carvalho, C.; Marinho, A.; Leal, B.; Bettencourt, A.; Boleixa, D.; Almeida, I.; Farinha, F.; Costa, P.P.;
Vasconcelos, C.; Silva, B.M. Association between vitamin D receptor (VDR) gene polymorphisms and
systemic lupus erythematosus in Portuguese patients. Lupus 2015, 24, 846–853. [CrossRef] [PubMed]
6. Abe, J.; Nakamura, K.; Takita, Y.; Nakano, T.; Irie, H.; Nishii, Y. Prevention of immunological disorders in
MRL/l mice by a new synthetic analogue of vitamin D3 : 22-oxa-1.ALPHA., 25-dihydroxyvitamin D3 . J. Nutr.
Sci. Vitaminol. 1990, 36, 21–31. [CrossRef] [PubMed]
7. Lemire, J.; Ince, A.; Takashima, M. 1,25-Dihydroxyvitamin D3 attenuates the expression of experimental
murine lupus of MRL/1 mice. Autoimmunity 1992, 12, 143–148. [CrossRef] [PubMed]
8. Koizumi, T.; Nakao, Y.; Matsui, T.; Nakagawa, T.; Matsuda, S.; Komoriya, K.; Kanai, Y.; Fujita, T. Effects of
Corticosteroid and 1,24R-Dihydroxy-Vitamin D3 Administration on Lymphoproliferation and Autoimmune
Disease in MRL/MP-lpr/lpr Mice. Int. Arch. Allergy Immunol. 1985, 77, 396–404. [CrossRef]
9. Yamamoto, E.; Jørgensen, T.N. Immunological effects of vitamin D and their relations to autoimmunity. J.
Autoimmun. 2019, 100, 7–16. [CrossRef]
10. Costenbader, K.H.; Feskanich, D.; Holmes, M.; Karlson, E.W.; Benito-Garcia, E. Vitamin D intake and risks of
systemic lupus erythematosus and rheumatoid arthritis in women. Ann. Rheum. Dis. 2008, 67, 530–535.
[CrossRef]
11. Ruiz-Irastorza, G.; Egurbide, M.V.; Olivares, N.; Martinez-Berriotxoa, A.; Aguirre, C. Vitamin D deficiency in
systemic lupus erythematosus: Prevalence, predictors and clinical consequences. Rheumatology 2008, 47,
920–923. [CrossRef]
12. Petri, M.; Bello, K.J.; Fang, H.; Magder, L.S. Vitamin D in systemic lupus erythematosus: Modest association
with disease activity and the urine protein-to-creatinine ratio. Arthritis Rheum. 2013, 65, 1865–1871. [CrossRef]
13. Abou-Raya, A.; Abou-Raya, S.; Helmii, M. The effect of vitamin D supplementation on inflammatory and
hemostatic markers and disease activity in patients with Systemic Lupus Erythematosus: A randomized
placebo-controlled trial. J. Rheumatol. 2013, 40, 265–272. [CrossRef]
14. Palmer, M.T.; Lee, Y.K.; Maynard, C.L.; Oliver, J.R.; Bikle, D.D.; Jetten, A.M.; Weaver, C.T. Lineage-specific
effects of 1,25-dihydroxyvitamin D3 on the development of effector CD4 T cells. J. Biol. Chem. 2011, 286,
997–1004. [CrossRef]
15. Kang, S.W.; Kim, S.H.; Lee, N.; Lee, W.-W.; Hwang, K.-A.; Shin, M.S.; Lee, S.-H.; Kim, W.-U.; Kang, I.
1,25-Dihyroxyvitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its
conserved noncoding sequence region. J. Immunol. 2012, 188, 5276–5282. [CrossRef]
16. Adzemovic, M.Z.; Zeitelhofer, M.; Hochmeister, S.; Gustafsson, S.A.; Jagodic, M. Efficacy of vitamin D in
treating multiple sclerosis-like neuroinflammation depends on developmental stage. Exp. Neurol. 2013, 249,
39–48. [CrossRef]
17. Drozdenko, G.; Heine, G.; Worm, M. Oral vitamin D increases the frequencies of CD38 + human B cells and
ameliorates IL-17-producing T cells. Exp. Dermatol. 2014, 23, 107–112. [CrossRef]
18. Chen, S.; Sims, G.P.; Chen, X.X.; Gu, Y.Y.; Chen, S.; Lipsky, P.E. Modulatory Effects of 1,25-Dihydroxyvitamin
D3 on Human B Cell Differentiation. J. Immunol. 2007, 179, 1634–1647. [CrossRef]
19. Haas, J.; Schwarz, A.; Korporal-Kuhnke, M.; Faller, S.; Jarius, S.; Wildemann, B. Hypovitaminosis D upscales
B-cell immunoreactivity in multiple sclerosis. J. Neuroimmunol. 2016, 294, 18–26. [CrossRef]
20. Chen, W.C.; Vayuvegula, B.; Gupta, S. 1,25-Dihydroxyvitamin D3-mediated inhibition of human B cell
differentiation. Clin. Exp. Immunol. 1987, 69, 639–646.
21. Heine, G.; Anton, K.; Henz, B.M.; Worm, M. 1alpha,25-dihydroxyvitamin D3 inhibits anti-CD40 plus
IL-4-mediated IgE production in vitro. Eur. J. Immunol. 2002, 32, 3395–3404. [PubMed]
22. Hartmann, B.; Heine, G.; Babina, M.; Steinmeyer, A.; Zügel, U.; Radbruch, A.; Worm, M. Targeting the vitamin
D receptor inhibits the B cell-dependent allergic immune response. Allergy 2011, 66, 540–548. [CrossRef]
[PubMed]
23. Qian, Y.; Qin, J.; Cui, G.; Naramura, M.; Snow, E.; Ware, C.F.; Fairchild, R.L.; Omori, S.A.; Rickert, R.C.;
Scott, M.; et al. Act1, a Negative Regulator in CD40- and BAFF-Mediated B Cell Survival. Immunity 2004, 21,
575–587. [CrossRef] [PubMed]
24. Qian, Y.; Giltiay, N.; Xiao, J.; Wang, Y.; Qin, J.; Tian, J.; Shuhua, H.; Scott, M.; Carter, R.; Li, X. Deficiency of
Act1, a critical modulator of B cell function, leads to development of Sjogren’s syndrome 1. Eur. J. Immunol.
2008, 38, 2219–2228. [CrossRef]
Nutrients 2020, 12, 291 16 of 17

25. Giltiay, N.V.; Lu, Y.; Allman, D.; Jørgensen, T.N.; Li, X. The adaptor molecule Act1 regulates BAFF
responsiveness and self-reactive B cell selection during transitional B cell maturation. J. Immunol. 2010, 185,
99–109. [CrossRef]
26. Giltiay, N.V.; Lu, Y.; Cullen, J.L.; Trine, N.; Shlomchik, M.J.; Li, X. Spontaneous loss of tolerance of autoreactive
B cells in Act1-deficient rheumatoid factor transgenic mice. J. Immunol. 2013, 191, 2155–2163. [CrossRef]
27. Harris, P.A.; Taylor, R.; Thielke, R.; Payne, J.; Gonzalez, N.; Conde, J.G. Research electronic data capture
(REDCap)—A metadata-driven methodology and workflow process for providing translational research
informatics support. J. Biomed. Inform. 2009, 42, 377–381. [CrossRef]
28. Jørgensen, T.N.; Thurman, J.; Izui, S.; Falta, M.T.; Metzger, E.T.; Flannery, A.S.; Kappler, J.; Marrack, P.;
Kotzin, B.L. Genetic susceptibility to PolyI:C-induced IFNα/β-dependent accelerated disease in lupus-prone
mice. Genes Immun. 2006, 7, 555–567. [CrossRef]
29. Smith, J.E.; Goodman, D.S. The turnover and transport of vitamin D and of a polar metabolite with the
properties of 25-hydroxycholecalciferol in human plasma. J. Clin. Investig. 1971, 50, 2159–2167. [CrossRef]
30. Gray, R.W.; Caldas, A.E.; Wilz, D.R.; Lemann, J.; Smith, G.A.; DeLuca, H.F. Metabolism and Excretion of 3
H-1,2 5-(OH)2 -Vitamin D3 in Healthy Adults. J. Clin. Endocrinol. Metab. 1978, 46, 756–765. [CrossRef]
31. Seeman, E.; Kumar, R.; Hunder, G.G.; Scott, M.; Heath, H.; Riggs, B.L. Production, degradation, and
circulating levels of 1,25-dihydroxyvitamin D in health and in chronic glucocorticoid excess. J. Clin. Investig.
1980, 66, 664–669. [CrossRef] [PubMed]
32. Wang, C.; Wu, L.; Bulek, K.; Martin, B.N.; Zepp, J.A.; Kang, Z.; Liu, C.; Herjan, T.; Misra, S.; Carman, J.A.;
et al. The psoriasis-associated D10N variant of the adaptor Act1 with impaired regulation by the molecular
chaperone hsp90. Nat. Immunol. 2013, 14, 72–81. [CrossRef] [PubMed]
33. Wu, L.; Wang, C.; Boisson, B.; Misra, S.; Rayman, P.; Finke, J.H.; Puel, A.; Casanova, J.-L.; Li, X. The differential
regulation of human ACT1 isoforms by Hsp90 in IL-17 signaling. J. Immunol. 2014, 193, 1590–1599. [CrossRef]
[PubMed]
34. Zhang, C.-J.; Wang, C.; Jiang, M.; Gu, C.; Xiao, J.; Chen, X.; Martin, B.N.; Tang, F.; Yamamoto, E.; Xian, Y.;
et al. Act1 is a negative regulator in T and B cells via direct inhibition of STAT3. Nat. Commun. 2018, 9, 2745.
[CrossRef]
35. Waddell, A.; Zhao, J.; Cantorna, M.T. NKT cells can help mediate the protective effects of
1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis in mice. Int. Immunol. 2015, 27,
237–244. [CrossRef]
36. Joshi, S.; Pantalena, L.-C.; Liu, X.K.; Gaffen, S.L.; Liu, H.; Rohowsky-Kochan, C.; Ichiyama, K.; Yoshimura, A.;
Steinman, L.; Christakos, S.; et al. 1,25-Dihydroxyvitamin D3 Ameliorates Th17 Autoimmunity via
Transcriptional Modulation of Interleukin-17A. Mol. Cell. Biol. 2011, 31, 3653–3669. [CrossRef]
37. Ikeda, U.; Wakita, D.; Ohkuri, T.; Chamoto, K.; Kitamura, H.; Iwakura, Y.; Nishimura, T.
1α,25-Dihydroxyvitamin D3 and all-trans retinoic acid synergistically inhibit the differentiation and expansion
of Th17 cells. Immunol. Lett. 2010, 134, 7–16. [CrossRef]
38. Johnson, A.C.; Davison, L.M.; Giltiay, N.V.; Vareechon, C.; Li, X.; Jørgensen, T.N. Lack of T cells in
Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease. Eur. J.
Immunol. 2012, 42, 1695–1705. [CrossRef]
39. Bhattacharya, D.; Cheah, M.T.; Franco, C.B.; Hosen, N.; Pin, C.L.; Sha, W.C.; Weissman, I.L. Transcriptional
Profiling of Antigen-Dependent Murine B Cell Differentiation and Memory Formation. J. Immunol. 2007, 179,
6808–6819. [CrossRef]
40. Jones, G.W.; McLoughlin, R.M.; Hammond, V.J.; Parker, C.R.; Williams, J.D.; Malhotra, R.; Scheller, J.;
Williams, A.S.; Rose-John, S.; Topley, N.; et al. Loss of CD4+ T cell IL-6R expression during inflammation
underlines a role for IL-6 trans signaling in the local maintenance of Th17 cells. J. Immunol. 2010, 184,
2130–2139. [CrossRef]
41. McFarland-Mancini, M.M.; Funk, H.M.; Paluch, A.M.; Zhou, M.; Giridhar, P.V.; Mercer, C.A.; Kozma, S.C.;
Drew, A.F. Differences in Wound Healing in Mice with Deficiency of IL-6 versus IL-6 Receptor. J. Immunol.
2010, 184, 7219–7228. [CrossRef]
42. Crotty, S. Follicular Helper CD4 T Cells (TFH ). Annu. Rev. Immunol. 2011, 29, 621–663. [CrossRef]
43. Kaji, T.; Hijikata, A.; Ishige, A.; Kitami, T.; Watanabe, T.; Ohara, O.; Yanaka, N.; Okada, M.; Shimoda, M.;
Taniguchi, M.; et al. CD4 memory T cells develop and acquire functional competence by sequential cognate
interactions and stepwise gene regulation. Int. Immunol. 2016, 28, 267–282. [CrossRef]
Nutrients 2020, 12, 291 17 of 17

44. Saxena, A.; Khosraviani, S.; Noel, S.; Mohan, D.; Donner, T.; Hamad, A.R.A. Interleukin-10 paradox: A potent
immunoregulatory cytokine that has been difficult to harness for immunotherapy. Cytokine 2015, 74, 27–34.
[CrossRef] [PubMed]
45. Guthmiller, J.J.; Graham, A.C.; Zander, R.A.; Pope, R.L.; Butler, N.S. Cutting Edge: IL-10 is essential for the
generation of germinal center B cell responses and anti-plasmodium humoral immunity. J. Immunol. 2017,
198, 617–622. [CrossRef]
46. Vaisberg, M.W.; Kaneno, R.; Franco, M.F.; Mendes, N.F. Influence of cholecalciferol (vitamin D3) on the
course of experimental systemic lupus erythematosus in F1 (NZBxW) mice. J. Clin. Lab. Anal. 2000, 14,
91–96. [CrossRef]
47. Briere, F. Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1
and IgG3. J. Exp. Med. 1994, 179, 757–762. [CrossRef]
48. Ishida, H. Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in
NZB/W F1 mice. J. Exp. Med. 1994, 179, 305–310. [CrossRef]
49. Ravirajan, C.T.; Wang, Y.; Matis, L.A.; Papadaki, L.; Griffiths, M.H.; Latchman, D.S.; Isenberg, D.A. Effect of
neutralizing antibodies to IL-10 and C5 on the renal damage caused by a pathogenic human anti-dsDNA
antibody. Rheumatology 2004, 43, 442–447. [CrossRef]
50. Park, Y.B.; Lee, S.K.; Kim, D.S.; Lee, J.; Lee, C.H.; Song, C.H. Elevated interleukin-10 levels correlated with
disease activity in systemic lupus erythematosus. Clin. Exp. Rheumatol. 1998, 16, 283–288.
51. Hedrich, C.M.; Rauen, T.; Apostolidis, S.A.; Grammatikos, A.P.; Rodriguez, N.R.; Ioannidis, C.; Kyttaris, V.C.;
Crispín, J.C.; Tsokos, G.C. Stat3 promotes IL-10 expression in lupus T cells through trans-activation and
chromatin remodeling. Proc. Natl. Acad. Sci. USA 2014, 111, 13457–13462. [CrossRef]
52. Enghard, P.; Langnickel, D.; Riemekasten, G. T cell cytokine imbalance towards production of IFN-γ and
IL-10 in NZB/W F1 lupus-prone mice is associated with autoantibody levels and nephritis. Scand. J. Rheumatol.
2006, 35, 209–216. [CrossRef] [PubMed]
53. Franco, A.S.; Freitas, T.Q.; Bernardo, W.M.; Pereira, R.M.R. Vitamin D supplementation and disease activity
in patients with immune-mediated rheumatic diseases: A systematic review and meta-analysis. Medicine
2017, 96, e7024. [CrossRef] [PubMed]
54. Jeffery, L.E.; Burke, F.; Mura, M.; Zheng, Y.; Qureshi, O.S.; Hewison, M.; Walker, L.S.K.; Lammas, D.A.; Raza, K.;
Sansom, D.M. 1,25-Dihydroxyvitamin D3 and IL-2 combine to inhibit T cell production of inflammatory
cytokines and promote development of regulatory T cells expressing CTLA-4 and FoxP3. J. Immunol. 2009,
183, 5458–5467. [CrossRef]
55. Chang, J.-H.; Cha, H.-R.; Lee, D.-S.; Seo, K.Y.; Kweon, M.-N. 1,25-Dihydroxyvitamin D3 inhibits the
differentiation and migration of T(H)17 cells to protect against experimental autoimmune encephalomyelitis.
PLoS ONE 2010, 5, e12925. [CrossRef]
56. Nakayama, Y.; Stabach, P.; Maher, S.E.; Mahajan, M.C.; Masiar, P.; Liao, C.; Zhang, X.; Ye, Z.-J.; Tuck, D.;
Bothwell, A.L.; et al. A limited number of genes are involved in the differentiation of germinal center B cells.
J. Cell. Biochem. 2006, 99, 1308–1325. [CrossRef] [PubMed]
57. Defrance, T.; Taillardet, M.; Genestier, L. T cell-independent B cell memory. Curr. Opin. Immunol. 2011, 23,
330–336. [CrossRef]
58. Obukhanych, T.V.; Nussenzweig, M.C. T-independent type II immune responses generate memory B cells. J.
Exp. Med. 2006, 203, 305–310. [CrossRef]
59. Snapper, C.M.; McIntyre, T.M.; Mandler, R.; Pecanha, L.M.; Finkelman, F.D.; Lees, A.; Mond, J.J. Induction
of IgG3 secretion by interferon gamma: A model for T cell- independent class switching in response to T
cell-independent type 2 antigens. J. Exp. Med. 1992, 175, 1367–1371. [CrossRef]
60. DeLuca, H.F.; Cantorna, M.T. Vitamin D: Its role and uses in immunology. FASEB J. 2001, 15, 2579–2585.
[CrossRef]
61. Tebben, P.J.; Singh, R.J.; Kumar, R. Vitamin D-Mediated Hypercalcemia: Mechanisms, Diagnosis, and
Treatment. Endocr. Rev. 2016, 37, 521–547. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).