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Interferon regulatory factor 5 is critical for the development of lupus in MRLlpr mice.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 3, March 2011, pp 738–748
DOI 10.1002/art.30183
© 2011, American College of Rheumatology
Interferon Regulatory Factor 5 Is Critical for the
Development of Lupus in MRL/lpr Mice
Yoshifumi Tada,1 Seiji Kondo,2 Shigehisa Aoki,1 Syuichi Koarada,1 Hisako Inoue,1
Rie Suematsu,1 Akihide Ohta,1 Tak W. Mak,3 and Kohei Nagasawa1
Objective. Interferon regulatory factor 5 (IRF-5)
is a transcription factor that mediates intracellular
signals activated by engagement of Toll-like receptors
(TLRs). IRF5 polymorphisms are associated with an
increased or decreased risk of systemic lupus erythematosus (SLE) in various human populations, but the
precise role of IRF5 in SLE development is not fully
understood. This study was undertaken to examine the
role of IRF5 in the development of murine lupus.
Methods. We crossed gene-targeted IRF5deficient (IRF5ⴚ/ⴚ) mice with MRL/MpJ-lpr/lpr (MRL/
lpr) mice and examined the progeny for survival, glomerulonephritis, autoantibody levels, immune system cell
populations, and dendritic cell function.
Results. IRF5ⴚ/ⴚMRL/lpr mice survived longer
than control IRF5ⴙ/ⴙMRL/lpr mice and displayed only
very mild glomerulonephritis. Autoantibodies to SLErelated nuclear antigens were lower in IRF5ⴚ/ⴚMRL/lpr
mouse serum, and numbers of activated CD4ⴙ T cells
were reduced in the spleen. Splenic DCs from IRF5ⴚ/ⴚ
MRL/lpr mice produced lower levels of inflammatory
cytokines when treated in vitro with TLR-7 or TLR-9
ligands or immune complexes. Interferon-␣ production
in response to CpG was also decreased.
Conclusion. Our results show that IRF5 is a
crucial driver of lupus development in mice, and indicate that IRF5 may be an attractive new target for
therapeutic intervention to control disease in SLE
patients.
Systemic lupus erythematosus (SLE) is a systemic
inflammatory autoimmune disease in humans that affects multiple organs and is characterized by production
of autoantibodies. These autoantibodies are mainly directed against nuclear antigens such as double-stranded
DNA (dsDNA), or against RNA-containing proteins
such as the Sm antigen or RNP (1). Attack by these
autoantibodies and immune cells results in damage to
multiple organs, including the kidney, skin, joints, central nervous system (CNS), and vascular system. The
damage to the kidney in SLE takes the form of glomerulonephritis characterized by the infiltration of activated
inflammatory cells and deposition of immune complexes (ICs) (2). The mechanism driving SLE is not yet
fully understood, but various studies have suggested that
type I interferon (IFN) plays a role in its pathogenesis.
Serum type I IFN levels are elevated in SLE patients
(3,4), and the expression of IFN-responsive genes is
elevated in peripheral blood cells from these individuals
(5,6). Furthermore, the frequencies of nephritis, hematopoietic disorders, and CNS disease are highest in SLE
patients whose peripheral blood cells show the greatest
increases in expression of IFN-responsive genes (5,6).
Recent genetic studies of SLE patients have
revealed that IRF5 gene polymorphisms are associated
with an increased risk of SLE (7–12). IRF5 is a member
of the IRF family of transcription factors that induce the
expression of IFN-responsive genes. Expression of the
IRF proteins is triggered by viral infection, IFNs, cytokines, and Toll-like receptor (TLR) ligands (13). Upon
stimulation of cells by viruses or TLR ligands, IRF5 is
activated and translocates to the nucleus, where it
Supported by the Ministry of Education, Culture, Sports,
Science, and Technology of Japan (grant 22590893) and by a research
project grant from the Faculty of Medicine, Saga University.
1
Yoshifumi Tada, MD, PhD, Shigehisa Aoki, MD, PhD,
Syuichi Koarada, MD, PhD, Hisako Inoue, MD, PhD, Rie Suematsu,
MD, Akihide Ohta, MD, PhD, Kohei Nagasawa MD, PhD: Saga
University, Saga, Japan; 2Seiji Kondo, MD, PhD: National Hospital
Organization Kyushu Medical Center, Fukuoka, Japan; 3Tak W. Mak,
PhD: Campbell Family Institute for Breast Cancer Research at
Princess Margaret Hospital and University Health Network, Toronto,
Ontario, Canada.
Drs. Tada and Kondo contributed equally to this work.
Address correspondence to Yoshifumi Tada, MD, PhD,
Department of Rheumatology, Faculty of Medicine, Saga University,
5-1-1 Nabeshima, Saga 849-8501, Japan. E-mail: taday@cc.saga-u.ac.jp.
Submitted for publication May 20, 2010; accepted in revised
form November 30, 2010.
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AMELIORATION OF LUPUS IN IRF5-DEFICIENT MRL/lpr MICE
induces the expression of IFN and chemokine genes
(14,15). Studies of IRF5⫺/⫺ mice have demonstrated
that IRF5 is critically involved in the production of
proinflammatory cytokines, particularly tumor necrosis
factor ␣ (TNF␣) and interleukin-6 (IL-6), by dendritic
cells (DCs) or macrophages treated with ligands of
TLR-3, TLR-4, TLR-7, or TLR-9 (16,17).
Although it is widely accepted that IRF5 is an
important genetic factor in the development of SLE
(18), its actual contribution to the development of lupus
or other forms of autoimmunity is unclear. To address
this issue, we investigated the role of IRF5 in a murine
model of SLE, the MRL/lpr strain. These mutants
spontaneously develop a systemic autoimmune disorder
(murine lupus) that closely resembles SLE in humans.
The clinical features of murine lupus include autoantibody production, hypergammaglobulinemia, glomerulonephritis, arthritis, vasculitis, splenomegaly, and lymphadenopathy (19–21). The molecular defect underlying
this phenotype is a mutation in the gene encoding Fas, a
cell surface receptor that triggers apoptosis (22,23).
In this study, we generated IRF5-deficient MRL/lpr
mice and analyzed their phenotype. In IRF5⫺/⫺MRL/lpr
mice, autoantibody production was markedly reduced,
glomerulonephritis was mild, and mouse survival was
much improved. Production of proinflammatory cytokines and IFN␣ by splenic DCs from IRF5⫺/⫺MRL/lpr
mice in response to treatment with TLR-7 and TLR-9
ligands or ICs was decreased. Our work demonstrates
that IRF5 is required for the development of murine
lupus in MRL/lpr mice, and supports the findings of
genetic studies indicating that IRF5 is an important
contributor to human SLE.
MATERIALS AND METHODS
Mice. Homozygous MRL/lpr mice were purchased
from Charles River Japan. Gene-targeted IRF5-deficient mice
(16) were backcrossed to MRL/lpr mice for ⬎8 generations
and genotyped by polymerase chain reaction (PCR) using DNA
obtained by tail biopsy. Survival analyses were performed using
female mice backcrossed for 8 generations, and other experiments were performed using male and female mice backcrossed for ⬎10 generations. Littermate IRF5⫹/⫹ mice were
used as controls. All mice were bred and maintained in the
Saga University animal facility, and all animal care was carried
out in accordance with institutional guidelines.
Measurement of autoantibodies and serum Ig. Antinuclear antibodies (ANAs) were detected by indirect immunofluorescence using HEp-2 cell slides (Orgentec) and
fluorescein-conjugated anti-mouse IgG (Santa Cruz Biotechnology). Sera were diluted at 1:100 and 1:500, and the fluorescence intensity of nuclear staining was scored on a scale of 0–3
by 2 independent observers in a blinded manner.
739
Anti-dsDNA antibodies were detected by enzymelinked immunosorbent assay (ELISA) as described previously
(24). Briefly, ELISA plates were coated with calf dsDNA and
blocked with phosphate buffered saline (PBS) containing 1%
bovine serum albumin (BSA). Mouse serum samples were then
serially diluted and added to the plates for a 1-hour incubation
at 37°C. Plates were washed, and peroxidase-conjugated goat
anti-mouse IgG1, IgG2a, IgG3, or IgM (Southern Biotechnology Associates) was added for 1 hour at 37°C. Twofold
dilutions of standard serum were added to each plate as an
internal control. A standard curve was constructed in which the
standard serum was defined as 100 arbitrary units, and the
antibody titers of experimental serum samples were calculated.
For measurement of anti-Sm and anti-RNP antibodies, ELISA
plates were precoated with purified Sm antigen or recombinant
human RNP (MBL). Primary antibody binding was detected
using peroxidase-conjugated goat anti-mouse IgG (Southern
Biotechnology Associates).
Mouse serum levels of IgG1, IgG2a, IgG3, and IgM
antibodies were measured by ELISA using a clonotyping system and a mouse Ig standard panel according to the recommendations of the manufacturer (Southern Biotechnology
Associates).
Histopathologic analysis, immunofluorescence, and
immunostaining. For histopathologic analysis, mouse kidneys
were fixed in buffered 10% formalin, embedded in paraffin,
sectioned, and stained with periodic acid–Schiff, periodic
acid–methenamine–silver (PAM), or hematoxylin and eosin.
Glomerular lesion severity (mesangial hypercellularity, proliferative changes, hyalinosis, exudates, necrosis, and/or crescent
formation) was scored on a scale of 0–3 by a pathologist (SA)
in a blinded manner as described previously (25). Perivascular
inflammation and tubular lesions (atrophy, dilation, and necrosis) were also scored in the same manner.
For immunofluorescence, mouse kidneys were embedded in TissueTek medium (Miles) and frozen in hexane
chilled in dry ice plus acetone. Sections were cut using a cryostat, fixed in acetone, rinsed with PBS, and incubated with
F(ab⬘)2 fluorescein-conjugated anti-mouse IgG (Santa Cruz
Biotechnology) or with F(ab⬘)2 fluorescein-conjugated antimouse C3 (MP Biomedicals). Glomerular deposition of IgG
and C3 was evaluated on a scale of 0–3 as described previously
(26).
For immunostaining, cryostat sections were incubated
with anti-CD4, anti-CD8, and anti-B220 rat anti-mouse monoclonal antibodies (mAb; BD Biosciences) to detect T cells, and
with anti-F4/80 rat anti-mouse mAb (Abcam) to detect macrophages. Antibodies were visualized using an immunoperoxidase method. Cellular infiltration in glomeruli, periglomeruli, and interstitial lesions was scored on a scale of 0–4,
where 0 ⫽ none, 1 ⫽ ⬍5 cells, 2 ⫽ 5–10 cells, 3 ⫽ 11–20 cells,
and 4 ⫽ ⬎21 cells per glomerulus or per 0.1 mm2. The mean ⫾
SEM scores for ⬎15 glomeruli or tissue areas were calculated.
Real-time quantitative reverse transcriptase–PCR
(RT-PCR). Levels of TNF␣, IL-6, IFN␥, and CCL5 (RANTES)
messenger RNA (mRNA) in mouse kidneys were measured by
real-time RT-PCR. Briefly, total RNA was extracted from the
cortex of kidneys that were snap-frozen in liquid nitrogen.
Quantitative PCR was performed using the SYBR Green
method and a LightCycler (Roche Diagnostics, Penzberg,
Germany). Primers were from Nihon Gene Research Labora-
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TADA ET AL
Purification of ICs. ICs were prepared from the sera of
aged MRL/lpr mice. Pooled sera were passed through a
0.22-␮m filter and applied to an antibody purification spin
column (Proteus Protein-G Kit; Prochem). Eluted IgG were
desalted, and ICs were isolated and concentrated using a
centrifugal filter device with a 300,000 molecular weight cutoff
(Pall) and dialyzed against DMEM. Anti-dsDNA antibody
Figure 1. Survival curves for female IRF5⫹/⫹MRL/lpr mice (n ⫽ 10),
IRF5⫹/⫺MRL/lpr mice (n ⫽ 26), and IRF5⫺/⫺MRL/lpr mice (n ⫽ 16),
determined by the Kaplan-Meier method. Loss of IRF5 prolonged the
survival of MRL/lpr mice.
tories. Relative mRNA expression was determined and normalized to the expression of the internal housekeeping gene
␤-actin.
Flow cytometric analysis. Mouse spleen cells were
suspended in PBS containing 1% BSA and 0.1% sodium azide
and incubated with various combinations of conjugated antibodies as described previously (24). Anti-CD4, anti-CD8, antiCD11c, anti-CD11b, anti-CD44, and anti-CD62L mAb were
purchased from BD Bioscience; anti-CD80 and anti-CD86
mAb were obtained from Caltag; anti-B220 and anti-CD3␧
mAb were from Beckman Coulter; and anti–PDCA-1 mAb was
from Miltenyi Biotec. FoxP3 staining was performed using a kit
(eBioscience). At least 3 ⫻ 105 immunostained cells per
sample were collected using a FACSCalibur and analyzed with
CellQuest software (BD Biosciences).
Analysis of DC cytokine production. CD11c⫹ DCs
were isolated from total mouse spleen cells by magnetic cell
sorting using anti-CD11c microbeads (Miltenyi Biotec).
Briefly, spleens from 2–3 mice/group (10–12 weeks old) were
digested with collagenase D (Roche Diagnostics). Mouse
spleen cells were blocked by incubation with anti-CD16/
32 mAb, labeled with anti-CD11c beads, and applied to a
magnetic separation column. After washing, the CD11c⫹ DCs
were eluted from the column by flushing. The resulting cell
purity was ⬎90% as determined by flow cytometry. Conventional DCs and plasmacytoid DCs (PDCs) were identified by
staining with anti-CD11c plus anti-B220 or anti–PDCA-1,
respectively.
For DC stimulation, DCs were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf
serum, seeded in 96-well plates at 3 ⫻ 105/well, and stimulated
for 24 hours with either TLR ligands or ICs. TLR ligands
(poly[I-C], lipopolysaccharide, flagellin, loxoribine, oligodeoxynucleotide (ODN) 1585, and ODN 1826) were all purchased
from InvivoGen, and ICs were isolated from mouse serum as
described below. Cytokines secreted into culture supernatants
were measured using a cytometric bead assay system (BD
Bioscience) and a FACSCalibur instrument. IFN␣ levels were
measured using an ELISA kit (PBL Biomedical).
Figure 2. Decreased serum autoantibody and Ig levels in IRF5⫺/⫺
MRL/lpr mice. A, Detection of antinuclear antibodies (ANAs) by
immunofluorescence. Serum samples (1:100 dilution) from 18-weekold IRF5⫹/⫹MRL/lpr or IRF5⫺/⫺MRL/lpr mice were applied to HEp-2
cell–bearing slides, and the binding of ANAs was detected by indirect
immunofluorescence. Representative stainings are shown. Original
magnification ⫻ 400. B, Fluorescence staining intensity scores. The
slides described in A were scored for the intensity of fluorescence
staining of sera (1:100 or 1:500 dilutions), as described in Materials
and Methods. C and D, Levels of anti–double-stranded DNA (antidsDNA) (C) and IgG anti-Sm and IgG anti-RNP (D) antibodies (Ab)
in IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mouse serum, detected by
enzyme-linked immunosorbent assay. Horizontal lines in B–D show
the median. E, Serum levels of total IgG1, IgG2a, IgG3, and IgM in
18-week-old IRF5⫹/⫹MRL/lpr mice (n ⫽ 12) and IRF5⫺/⫺MRL/lpr
mice (n ⫽ 11). Bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.01; ⴱⴱ ⫽ P ⬍
0.005; ⴱⴱⴱ ⫽ P ⬍ 0.001, by Mann-Whitney U test in B–D and by
Student’s t-test in E. AU ⫽ arbitrary units.
AMELIORATION OF LUPUS IN IRF5-DEFICIENT MRL/lpr MICE
741
Figure 3. Amelioration of glomerulonephritis in IRF5⫺/⫺MRL/lpr mice. Kidneys from 18-week-old IRF5⫹/⫹
MRL/lpr mice (n ⫽ 13) and IRF5⫺/⫺MRL/lpr mice (n ⫽ 12) were examined for renal disease. A, Representative
images of glomerular lesions from IRF5⫹/⫹MRL/lpr mice (i–iii) and IRF5⫺/⫺MRL/lpr mice (iv–vi), stained with
periodic acid–Schiff (PAS) (i and iv), periodic acid–methenamine–silver (PAM) (ii and v), or hematoxylin and
eosin (H&E) (iii and vi). Original magnification ⫻ 200 in i, ii, iv, and v; ⫻ 40 in iii and vi. B, Glomerular, vascular,
and tubular disease scores. The slides described in A were scored on a scale of 0–3. C, Serum blood urea nitrogen (BUN) levels in 18-week-old IRF5⫹/⫹MRL/lpr mice (n ⫽ 12) and IRF5⫺/⫺MRL/lpr mice (n ⫽ 11).
D, Deposition of IgG and C3 in the glomeruli of IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice at 18 weeks of age.
Representative images are shown. Original magnification ⫻ 100. E, Staining intensity scores for IgG and C3
(n ⫽ 7 mice per group). Bars show the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.005; ⴱⴱⴱ ⫽ P ⬍ 0.001;
ⴱⴱⴱⴱ ⫽ P ⬍ 0.0001, by Student’s t-test.
levels and IgG concentrations of purified ICs were determined
by ELISA.
Statistical analysis. Statistical analyses were performed using GraphPad Prism 5.01 software. In most experiments, Student’s unpaired 2-tailed t-test was used to determine
the statistical significance of differences between groups. The
Mann-Whitney U test was used for statistical analysis of differences in autoantibody levels, cytokine mRNA levels measured by RT-PCR, and renal immunostaining scores. Mouse
survival was analyzed by the Kaplan-Meier method and the log
rank test.
RESULTS
Prolonged survival of IRF5-deficient MRL/lpr
mice. We first determined whether loss of IRF5 would
have any effect on the survival of female MRL/lpr mice.
IRF5⫺/⫺MRL/lpr mice lived much longer than IRF5⫹/⫹
MRL/lpr littermate controls (Figure 1). All IRF5⫹/⫹
MRL/lpr mice died by 52 weeks of age, whereas only
12.5% of the IRF5⫺/⫺MRL/lpr mice had died by that
age. This effect on survival was dose-dependent, as
evidenced by the finding that 50% of IRF5⫹/⫺MRL/lpr
mice lived longer than 52 weeks (P ⬍ 0.0001). These
results suggest that IRF5 promotes disease progression
and contributes to the premature death of MRL/lpr mice.
Decreased autoantibody levels in IRF5-deficient
MRL/lpr mice. We measured concentrations of autoantibodies and Ig in the sera of IRF5⫹/⫹MRL/lpr and
IRF5⫺/⫺MRL/lpr mice at 18 weeks of age. Whereas
ANA in the sera of IRF5⫹/⫹MRL/lpr mice was readily
detected by its strong fluorescence intensity, sera from
IRF5⫺/⫺MRL/lpr mice showed only weak ANA staining
(Figure 2A). This pattern held when sera were diluted to
1:100 or 1:500 (P ⬍ 0.001 and P ⬍ 0.005, respectively)
(Figure 2B). Serum levels of the IgG subclasses of antidsDNA antibodies were also significantly decreased in
IRF5⫺/⫺MRL/lpr mice compared to IRF5⫹/⫹MRL/lpr
mice (P ⬍ 0.01 for IgG1; P ⬍ 0.001 for IgG2a) (Figure
2C). The IgG3 subclass of anti-dsDNA antibodies was
undetectable in IRF5⫺/⫺MRL/lpr mice. In contrast,
levels of anti-dsDNA antibodies of the IgM subclass
were similar in IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr
mice. Notably, neither IgG anti-Sm antibodies nor IgG
anti-RNP antibodies were detectable in the sera of
IRF5⫺/⫺MRL/lpr mice (Figure 2D). With respect to serum
742
TADA ET AL
Figure 4. Decreased immune cell infiltration and production of inflammatory cytokines in the absence of IRF5.
Cell infiltration into the kidneys of 18-week-old IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice was examined.
A, Representative images of kidney tissue, showing glomerular or interstitial infiltration of CD4⫹, CD8⫹, and
B220⫹ T cells and F4/80⫹ macrophages. Arrows indicate positively stained cells. Original magnification ⫻ 400.
B, Scores for infiltration of CD4⫹, CD8⫹, and B220⫹ T cells and F4/80⫹ macrophages. Bars show the mean ⫾
SEM (n ⫽ 6 mice per group). C, Levels of mRNA for tumor necrosis factor ␣ (TNF␣), interleukin-6 (IL-6),
CCL5, and interferon-␥ (IFN␥) in the kidney cortex of IRF5⫹/⫹MRL/lpr mice (n ⫽ 5) and IRF5⫺/⫺MRL/lpr mice
(n ⫽ 4), assessed by reverse transcriptase–polymerase chain reaction. Bars show the mean ⫾ SEM ratio of
cytokine mRNA to ␤-actin mRNA. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.005, by Mann-Whitney U test.
Ig, IgG3 levels were significantly decreased in the absence of IRF5 (P ⬍ 0.001), but serum IgG1, IgG2a,
and IgM levels were comparable to those in controls
(Figure 2E). These data indicate that the production of
autoantibodies, including ANA, anti-dsDNA antibody,
anti-Sm antibody, and anti-RNP antibody, is dramatically attenuated in MRL/lpr mice deficient in IRF5.
Amelioration of glomerulonephritis in IRF5deficient MRL/lpr mice. To assess the effect of IRF5 on
autoimmune phenotypes in mouse organs, we scored the
pathologic severity of glomerular, vascular, and tubular
lesions in the kidneys of 18-week-old IRF5⫹/⫹MRL/
lpr and IRF5⫺/⫺MRL/lpr mice. IRF5⫹/⫹MRL/lpr mice
showed severe glomerulonephritis at this age, exhibiting
mesangial proliferation, necrosis, lobulation, and crescent formation in glomeruli (Figure 3A, part i). Both
destruction and neoformation of basement membranes
were detected in these control animals by PAM staining
(Figure 3A, part ii). In contrast, glomerular lesions in
IRF5⫺/⫺MRL/lpr mice were very mild, with only low to
moderate mesangial proliferation and almost intact
basement membranes (Figure 3A, parts iv and v).
Whereas typical dense perivascular infiltration of leukocytes was apparent in IRF5⫹/⫹MRL/lpr mice (Figure
3A, part iii), IRF5⫺/⫺ MRL/lpr mice showed minimal or
only mild infiltration around vessels (Figure 3A, part vi).
The total pathologic scores for the glomerular, vascular,
and tubular lesions in IRF5⫺/⫺MRL/lpr mice were significantly lower than those in IRF5⫹/⫹MRL/lpr controls (P ⬍ 0.001, P ⬍ 0.0001, and P ⬍ 0.05, respectively)
(Figure 3B). Moreover, serum blood urea nitrogen
concentration, an indicator of renal function, was significantly reduced in IRF5⫺/⫺MRL/lpr mice at 18 weeks of
age compared to IRF5⫹/⫹MRL/lpr mice (mean ⫾ SEM
29.7 ⫾ 5.4 mg/dl versus 40.2 ⫾ 7.9 mg/dl; P ⬍ 0.005)
(Figure 3C).
Deposition of IgG and complement component
C3 in glomeruli is a characteristic feature of the glomerulonephritis observed in MRL/lpr mice. However, in
contrast to the intense deposition of IgG and C3 in the
glomeruli of IRF5⫹/⫹MRL/lpr mice, histologic analysis
of the glomeruli of IRF5⫺/⫺MRL/lpr mice revealed only
mild to moderate deposition of IgG, and no or very little
C3 deposition (Figure 3D). Accordingly, the glomerular
deposition scores for IgG and C3 were significantly
decreased in IRF5⫺/⫺MRL/lpr mice compared to
IRF5⫹/⫹MRL/lpr mice (P ⬍ 0.05 and P ⬍ 0.001, respectively) (Figure 3E).
AMELIORATION OF LUPUS IN IRF5-DEFICIENT MRL/lpr MICE
743
Figure 5. Altered splenic lymphocyte and dendritic cell (DC) populations in the absence of IRF5. A, Decreased
splenomegaly and lymph node (LN) cellularity. Spleen weight (left) and the total cell numbers of axillary and
inguinal LNs (right) in 18-week-old IRF5⫹/⫹MRL/lpr mice (n ⫽ 13) and IRF5⫺/⫺MRL/lpr mice (n ⫽ 12) are
shown. Horizontal lines show the median. B, Levels of B220⫹CD3⫹, B220⫺CD3⫹, CD19⫹, CD4⫹, and CD8⫹
cells in IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice. Splenocytes were immunostained to detect the indicated
markers. C, Altered subpopulations of splenic CD4⫹ T cells. CD4⫹ T cells were fractionated into naive,
activated, memory, unique abnormal (B220⫹), and regulatory (FoxP3⫹) subsets. D, Levels of macrophages
(CD11b⫹CD11c⫹), conventional DCs (cDCs; CD11c⫹B220⫺), and plasmacytoid DCs (PDCs; CD11c⫹PDCA1⫹) in total splenocytes from IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice (n ⫽ 8 per group), detected by
immunostaining. E, Levels of CD80 and CD86 on conventional DCs and PDCs. RIgG ⫽ antibody binding
control. In B–D, bars show the mean ⫾ SEM percentage of total cells. In E, bars show the mean ⫾ SEM mean
fluorescence intensity (MFI). ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001; ⴱⴱⴱⴱ ⫽ P ⬍ 0.0001, by Student’s
t-test.
We next identified the types of cells infiltrating
the mouse kidney by immunostaining with anti-CD4,
anti-CD8, and anti-B220 mAb to detect T cells, and with
anti-F4/80 mAb to detect macrophages. As previously
reported, anti-B220–positive cells in the MRL/lpr mouse
kidney are unique double-negative T cells (27). The glomeruli of IRF5⫹/⫹MRL/lpr mice showed many infiltrating cells, which marker analysis identified as mainly CD4⫹
T cells and macrophages (Figure 4A, top). In contrast, there was very little inflammatory cell infiltration
in the glomeruli of IRF5⫺/⫺MRL/lpr mice (Figure 4A,
bottom). Analysis of cellular invasion into the glomeruli and the interstitial area showed that the infiltration
of CD4⫹ T cells and macrophages into the glomeruli
was significantly decreased in the absence of IRF5
(Figure 4B). These data indicate that IRF5 promotes
inflammatory cell infiltration in the kidneys of MRL/lpr
mice.
Previous work has shown that inflammatory cytokines and chemokines are up-regulated in the kidneys
of MRL/lpr mice (28,29). We therefore compared levels
of various cytokine transcripts in kidney cortices from
IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice. Levels of
TNF␣, IL-6, and CCL5 mRNA were significantly reduced in the kidneys of IRF5⫺/⫺MRL/lpr mice compared to controls (P ⬍ 0.05 for all), whereas IFN␥
expression was comparable in the kidneys of IRF5⫹/⫹
MRL/lpr and IRF5⫺/⫺MRL/lpr mice (Figure 4C). Thus,
IRF5 is important for inflammatory cytokine and chemokine expression. Taken together, these results indicate that the progression of glomerulonephritis in MRL/
lpr mice is mitigated in the absence of IRF5.
Altered lymphocyte and DC populations in the
spleens of IRF5-deficient MRL/lpr mice. MRL/lpr mice
exhibit autoimmune symptoms in various lymphoid organs, including splenomegaly. We therefore compared
spleen weights, lymph node (LN) cell numbers, and
splenic lymphocyte populations in IRF5⫹/⫹MRL/lpr and
IRF5⫺/⫺MRL/lpr mice. Splenomegaly was significantly
reduced in the absence of IRF5 (mean ⫾ SEM 0.59 ⫾
0.13 gm versus 0.82 ⫾ 0.28 gm; P ⬍ 0.05) (Figure 5A,
left). The total number of LN cells also tended to be
lower in IRF5⫺/⫺MRL/lpr mice although the difference
was not statistically significant (Figure 5A, right). Flow
cytometric analysis of total mouse spleen cell populations revealed that the numbers of CD4⫹ T cells
744
TADA ET AL
were significantly decreased (P ⬍ 0.01), but CD19⫹
B cells and CD8⫹ T cells were significantly increased
(P ⬍ 0.0001 and P ⬍ 0.001, respectively), in IRF5⫺/⫺
MRL/lpr mice (Figure 5B). Among CD4⫹ T cells, the
memory (CD62LlowCD44high; P ⬍ 0.05) and activated
(CD62LhighCD44high; P ⬍ 0.01) CD4⫹ T cell populations were decreased in the absence of IRF5, as were
B220⫹ activated CD4⫹ T cells (P ⬍ 0.05) (Figure 5C).
In contrast, the naive (CD62LhighCD44low) and regulatory (FoxP3⫹) CD4⫹ T cell subpopulations showed no
marked change (Figure 5C).
In addition to altered T cell populations, the
spleens of IRF5⫺/⫺MRL/lpr mice showed increases in
the percentages of CD11b⫹CD11c⫹ macrophages and
CD11c⫹PDCA-1⫹ PDCs (P ⬍ 0.01 and P ⬍ 0.05, respectively) (Figure 5D). However, the percentage of
CD11c⫹B220⫺ conventional DCs was similar to that in
spleens of IRF5⫹/⫹MRL/lpr mice. When we evaluated
the expression of the costimulatory molecules CD80 and
CD86 on DCs to assess their activation status, we found
that CD86 could be detected on both conventional DCs
and PDCs in both groups of mice whereas CD80 could
not, and that the expression levels of these molecules
were similar (Figure 5E). These data indicate that
activated CD4⫹ T cells were decreased in the absence of
IRF5 in MRL/lpr mice.
Defective cytokine production by DCs from IRF5deficient MRL/lpr mice in response to TLR-7 and TLR-9
engagement. Previous studies have shown that DCs from
IRF5⫺/⫺ mice produce decreased levels of IFNs and
other cytokines in response to treatment with TLR
ligands (16,17). We investigated whether loss of IRF5
had a similar effect on TLR-mediated cytokine production by splenic DCs from MRL/lpr mice. We first
evaluated the percentages of conventional DCs and
PDCs among splenic CD11c⫹ DCs purified from
Figure 6. Defective cytokine production by splenic dendritic cells (DCs) in the absence of IRF5. CD11c⫹ DCs
were purified from the spleens of 12–14-week-old IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice (n ⫽ 2–3 mice
per experiment). DCs were stimulated in vitro for 24 hours with Toll-like receptor (TLR) ligands or immune
complexes (ICs). A, Levels of tumor necrosis factor ␣ (TNF␣), interleukin-6 (IL-6), IL-10, interferon-␥ (IFN␥),
and monocyte chemotactic protein 1 (MCP-1) in mouse DCs stimulated with the TLR ligands poly(I-C) (10
␮g/ml), lipopolysaccharide (LPS; 1 ␮g/ml), flagellin (1 ␮g/ml), loxoribine (1 mM), and CpG (1 ␮M). Supernatants
were collected and concentrations of the indicated cytokines were measured by cytometric bead assay. B, Levels
of IFN␣ in mouse DCs stimulated with the TLR ligands poly(I-C) (10 ␮g/ml) and CpG (1 ␮M). Supernatants
were collected and concentrations of the indicated cytokines were measured by enzyme-linked immunosorbent
assay. C, Levels of TNF␣ and IL-6 in mouse DCs stimulated with ICs purified from IRF5⫹/⫹MRL/lpr mouse
serum. Supernatants were collected and concentrations of the indicated cytokines were measured by cytometric
bead assay. Bars show the mean ⫾ SEM cytokine concentrations from 5 experiments. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍
0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001, by Student’s t-test.
AMELIORATION OF LUPUS IN IRF5-DEFICIENT MRL/lpr MICE
IRF5⫹/⫹MRL/lpr and IRF5⫺/⫺MRL/lpr mice but found
no difference (for IRF5⫹/⫹MRL/lpr mice, mean ⫾ SEM
61.2 ⫾ 9.8% for conventional DCs and 29.9 ⫾ 8.9% for
PDCs; for IRF5⫺/⫺MRL/lpr mice, mean ⫾ SEM 58.7 ⫾
8.3% for conventional DCs and 32.4 ⫾ 6.9% for PDCs;
n ⫽ 5 mice per group). Levels of TNF␣, IL-6, and IL-10
production by splenic DCs from IRF5⫺/⫺MRL/lpr mice
were markedly decreased in response to the TLR-9
ligand CpG (type B) and TLR-7 ligand loxorbine (Figure 6A). Levels of IFN␥ and monocyte chemotactic
protein 1 were also reduced in response to CpG. Similarly, IFN␣ production by IRF5⫺/⫺MRL/lpr DCs in
response to treatment with CpG (type A) was decreased
(Figure 6B). Thus, as was the case in mice of a nonautoimmune background, IRF5 deficiency attenuated inflammatory cytokine and chemokine production in
MRL/lpr mice.
It has recently been demonstrated that ICs containing DNA or RNA can activate DCs and B cells via
engagement of TLRs and Fc␥ receptors (30–33). To
examine the impact of IRF5 loss on IC effects, we
purified ICs from the sera of MRL/lpr mice, applied
them to mouse splenic DCs, and measured cytokine
production. Although TNF␣ and IL-6 production by
splenic DCs from IRF5⫹/⫹MRL/lpr mice was enhanced
by stimulation with ICs in a dose-dependent manner,
IC-induced cytokine production was significantly reduced in DCs from IRF5⫺/⫺MRL/lpr mice (P ⬍ 0.05)
(Figure 6C). We did not detect IFN␣ production in
response to ICs from DCs of IRF5⫹/⫹MRL/lpr and
IRF5⫺/⫺MRL/lpr mice. Taken together, these data suggest that IRF5 promotes the activation of DCs that are
stimulated by ICs or by TLR-7 or TLR-9 engagement,
and that, in the absence of IRF5, there is a deficit in
proinflammatory cytokine production that decreases the
autoimmune symptoms of MRL/lpr mice.
DISCUSSION
Since 2005, many reports have been published
demonstrating an association between IRF5 polymorphisms and an increased or decreased risk of SLE in
humans (7–12). In contrast to the wealth of genetic
findings, little has been discovered about the actual role
of the IRF-5 protein in autoimmunity and the development of autoimmune diseases. In SLE patients, certain
IRF5 polymorphisms are associated with increased expression of IFN-responsive genes as well as with elevated serum IFN␣ levels (11,34). ICs from the serum of
SLE patients induced the production of IL-6 and type I
IFN by murine DCs, and this effect was abolished in
745
DCs lacking IRF5 or IRF7 (35). In this study, we
demonstrated that IRF5 deficiency reduced the production of autoantibodies and attenuated the development
of glomerulonephritis such that mouse survival was
greatly improved. Thus, we propose that IRF5 plays a
crucial role in the development of systemic autoimmune
disease in MRL/lpr mice.
IRF5 is required for the production of proinflammatory cytokines induced by TLR ligands (16). Although it was originally unexpectedly found that type I
IFN production was not decreased in IRF5⫺/⫺ mouse
PDCs (16), a subsequent study showed that IRF5 was
necessary for type I IFN production in response to
certain stimuli (17,36). In this study, we demonstrated
that TLR-7– and TLR-9–induced proinflammatory cytokine production and TLR-9–induced IFN␣ production
by mouse splenic DCs are regulated by IRF5. However,
our in vitro data show minor discrepancies with previous
reports. For example, TLR-3– and TLR-4–induced cytokine production was not affected in DCs lacking IRF5
in our study, but has been shown to be suppressed in
others (16,35). This discrepancy may be due to differences in cell populations examined, i.e., splenic DCs
versus bone marrow–derived DCs versus macrophages;
the genetic background of the mice used; or the sources
of the stimuli (17).
Our results suggest that loss of IRF5 downregulates DC activation signals delivered through TLR-7
and TLR-9, leading to attenuated disease in IRF5⫺/⫺
MRL/lpr mice. Previous studies have shown that TLR-7
and TLR-9 play significant roles in the development
of lupus in MRL/lpr mice (37–40). TLR-7–deficient
MRL/lpr mice showed milder glomerulonephritis and
decreased levels of anti-Sm and anti-RNP antibodies
(39), whereas TLR-9–deficient MRL/lpr mice had exacerbated glomerulonephritis and heightened lymphocyte
activation (37–39). Interestingly, however, TLR-7 and
TLR-9 double-knockout MRL/lpr mice showed a marked
reduction in autoantibodies and glomerulonephritis
(40), and 3d mutant B6-Faslpr mice, which have defective
TLR-3, TLR-7, and TLR-9 signaling, also showed reduced levels of autoantibodies and improved survival
(41). These data suggest that blockade of TLR-7 or
TLR-9 alone has a limited effect on autoantibody production and opposing effects in glomerulonephritis, but
the simultaneous blocking of both TLR-7 and TLR-9
abolishes most of the clinical features of murine lupus.
We found that disease in the IRF5-deficient
MRL/lpr mice was similar to that observed in MRL/lpr
mice lacking both TLR-7 and TLR-9 (40), although the
glomerulonephritis in the IRF5-deficient MRL/lpr mice
746
was milder and their survival longer. Disease in IRF5deficient MRL/lpr mice was also similar to that in 3d
mutant B6-Faslpr mice (41). These studies suggest that
IRF5 plays a key role in the development of lupus by
regulating signals through TLR-7 and TLR-9, an essential combination for full-blown lupus. It is possible that
another pathway mediated by IRF5 but not triggered by
TLR-7 or TLR-9 engagement contributes to the attenuated disease observed in IRF5⫺/⫺MRL/lpr mice. As
described previously, signals through TLR-3 or TLR-4
are candidate routes. Further study will clarify the
precise function of IRF5 in the activation of various
immune system cells in MRL/lpr mice.
It has been reported that ICs are important
stimuli that promote the activation of the TLR-7 and
TLR-9 pathways in murine lupus (30–33,35). DNAcontaining ICs activate DCs through TLR-9 and induce
the production of TNF␣, IL-12, and IFN␣ (30,31), and
RNA-containing ICs activate DCs through TLR-7 and
result in IL-6 and IFN␣ production (33,35). Our findings
confirmed that TNF␣ and IL-6 production by DCs was
enhanced in response to ICs purified from MRL/lpr
mice, and that this effect was diminished in DCs lacking
IRF5. These data suggest that IRF5 promotes lupus by
inducing these inflammatory cytokines, presumably in
response to ICs composed of autoantibodies and nucleic
acids. We also observed increased expression of TNF␣,
IL-6, and CCL5 in the kidney. It has been shown that
IL-6 plays important roles in lupus (42) and that TNF␣
promotes focal inflammation in murine lupus (43),
although genetic loss of Tnfr1 accelerates the progression of disease (44).
Previous studies of human SLE and mice with
lupus showed conflicting results with regard to the contribution of type I IFN to disease progression. In human
SLE, type I IFN is considered to be pathogenic and its
expression is linked to a susceptibility haplotype of IRF5
(5,6,34), whereas it has been reported that type I IFN
protects against murine lupus (45). Our data showing
decreased IFN␣ production in response to CpG from
IRF5-deficient DCs suggest that loss of IRF5 promotes
lupus. However, the effects of IRF5 in the induction of
type I IFNs vary based on cell type and/or stimulus
(16,17,36), and we did not detect IFN␣ production in
response to ICs. Although further study is necessary to
explore the type I IFN profile in these mice, we speculate that IRF5 is a more critical regulator of proinflammatory cytokines than type I IFNs, and/or that the
beneficial effects of reduced inflammatory cytokine levels overcome the effect of decreased type I IFN production. Irregardless, the pathogenic role of IRF5 in MRL/
TADA ET AL
lpr mice appears to be independent of type I IFN and
may not be equal to that in human SLE.
Recently, phenotypes of the lupus models
Fc␥RIIB⫺/⫺ Yaa and Fc␥RIIB⫺/⫺ mice (46), and mice
with pristane-induced lupus (47), all of which are IRF5deficient, have been described. These mice showed
diminished autoantibody production and attenuated
glomerulonephritis. Many of the results of these studies
are consistent with our data. However, lymphosplenomegaly and serum IgG levels were markedly reduced in
the IRF5-deficient Fc␥RIIB⫺/⫺ Yaa mice (46), whereas
IRF5⫺/⫺MRL/lpr mice showed only a modest decrease
in splenomegaly and LN cellularity, and only serum
IgG3 was decreased. Despite these differences, these
models are consistent in their delineation of a critical
role for IRF5 in murine lupus.
In conclusion, we confirmed in vivo that IRF5
deficiency prevents the development of autoantibody
production and glomerulonephritis in MRL/lpr mice,
and thereby extends mouse survival. We also confirmed
the findings of previous genetic studies showing that
IRF5 is critically involved in the pathogenesis of SLE.
Our results have important therapeutic implications;
namely, that treatment with inhibitors targeting IRF5
may be beneficial for SLE patients.
ACKNOWLEDGMENTS
We thank Motoko Fujisaki for technical assistance and
animal care, Yumiko Tsugitomi for technical help with the
histology, immunofluorescence, and immunostaining studies,
and Mary Saunders for scientific editing of this manuscript.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Tada had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Tada, Kondo, Koarada, Inoue,
Suematsu, Ohta, Nagasawa.
Acquisition of data. Tada, Kondo, Aoki, Koarada.
Analysis and interpretation of data. Tada, Kondo, Aoki, Mak.
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