Pleiotropic IFN-Dependent and -Independent
Effects of IRF5 on the Pathogenesis of
Experimental Lupus
This information is current as
of June 18, 2017.
Yuan Xu, Pui Y. Lee, Yi Li, Chao Liu, Haoyang Zhuang,
Shuhong Han, Dina C. Nacionales, Jason Weinstein, Clayton
E. Mathews, Lyle L. Moldawer, Shi-Wu Li, Minoru Satoh,
Li-Jun Yang and Westley H. Reeves
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Copyright © 2012 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:4113-4121; Prepublished online 14
March 2012;
doi: 10.4049/jimmunol.1103113
http://www.jimmunol.org/content/188/8/4113
The Journal of Immunology
Pleiotropic IFN-Dependent and -Independent Effects of IRF5
on the Pathogenesis of Experimental Lupus
Yuan Xu,* Pui Y. Lee,*,1 Yi Li,* Chao Liu,† Haoyang Zhuang,* Shuhong Han,*
Dina C. Nacionales,‡ Jason Weinstein,*,2 Clayton E. Mathews,† Lyle L. Moldawer,‡
Shi-Wu Li,† Minoru Satoh,*,† Li-Jun Yang,† and Westley H. Reeves*,†
T
he transcription factor IFN regulatory factor 5 (IRF5)
is a member of the IRF family with a key role in TLRstimulated production of proinflammatory cytokines such
as IL-12, IL-23, IL-6, and TNF-a (1), activation of type I IFN
genes (2, 3), regulation of apoptosis (4), and development of
B cells (5, 6). In humans, there are multiple IRF5 isoforms
resulting from alternative splicing of the IRF5 gene (7–10). In
contrast, murine IRF5 is expressed as a single transcript (11).
Certain genetic polymorphisms of IRF5 are strongly associated
with an increased risk of developing systemic lupus erythematosus
(SLE) in humans, and the IRF5 haplotype helps to define the risk
*Division of Rheumatology and Clinical Immunology, University of Florida, Gainesville, FL 32610; †Department of Pathology, Immunology and Laboratory Medicine,
University of Florida, Gainesville, FL 32610; and ‡Department of Surgery, University
of Florida, Gainesville, FL 32610
1
Current address: Children’s Hospital Boston, Boston, MA.
2
Current address: Yale University School of Medicine, New Haven, CT.
Received for publication October 31, 2011. Accepted for publication February 16,
2012.
This work was supported by Research Grant R01-AR44731 from the U.S. Public
Health Service and a grant from the Lupus Research Institute. H.Z. was a National
Institutes of Health T32 trainee (AR007603).
Address correspondence and reprint requests to Dr. Westley H. Reeves, Division of
Rheumatology and Clinical Immunology, University of Florida, P. O. Box 100221,
Gainesville, FL 32610-0221. E-mail address: [email protected]
for SLE (7, 10, 12–15). IRF5 also contributes to the pathogenesis
of lupus in mouse models. In the FcgRIIB2/2Yaa and FcgRIIB2/2
lupus models, IRF5 is required for autoantibody production and
renal disease (16). The mechanism appears to be partly independent of IFN-I production, but additional mechanisms have not
been defined. IRF5 deficiency also abolishes anti-Sm/RNP Abs
and reduces anti-dsDNA autoantibodies and inflammatory cytokine production while decreasing renal disease and improving
survival in MRL/lpr mice (17). Although IFN-I ameliorates lupus
in the MRL/lpr model (18), lupus induced by pristane is mediated
by signaling through the type I IFN receptor (IFNAR) and TLR7
(19). In a recent study, autoantibody production and renal disease
were abolished in pristane-treated IRF52/2 mice, an effect ascribed to a B cell-intrinsic IRF5 requirement for class switching to
IgG2a, the predominant autoantibody isotype in the pristane lupus
model (6). The present study was carried out to further define the
mechanisms by which IRF5 influences the development of autoimmune disease in mice. We present evidence that the effects of
IRF5 on the induction of autoimmune disease in pristane-treated
mice is more complex than previously believed, with IFNdependent or -independent effects on multiple cell lineages including B and T lymphocytes, monocyte/macrophages, and plasmacytoid dendritic cells (pDC).
Materials and Methods
The online version of this article contains supplemental material.
Mice and pristane treatment
Abbreviations used in this article: B6, C57BL/6; IFNAR, type I IFN receptor; IRAK,
IL-1R–associated kinase; IRF5, IFN regulatory factor 5; ISG, IFN-stimulated gene;
pDC, plasmacytoid dendritic cell; PDCA-1, plasmacytoid dendritic cell Ag-1; QPCR, quantitative PCR; SLE, systemic lupus erythematosus; TMPD, 2,6,10,14-tetramethylpentadecane.
Mice were bred and maintained under specific pathogen-free conditions at
the University of Florida Animal Facility. IRF52/2 mice on a C57BL/6
(B6) background were provided by Dr. Katherine Fitzgerald (University of
Massachusetts, Worcester, MA) with permission from Dr. Tak Mak
(University of Toronto, Toronto, ON, Canada) and were backcrossed to B6
for at least 10 generations. B6 MyD882/2 mice were provided by Dr. Lyle
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103113
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Genetic polymorphisms of IFN regulatory factor 5 (IRF5) are associated with an increased risk of lupus in humans. In this study, we
examined the role of IRF5 in the pathogenesis of pristane-induced lupus in mice. The pathological response to pristane in IRF52/2
mice shared many features with type I IFN receptor (IFNAR)2/2 and TLR72/2 mice: production of anti-Sm/RNP autoantibodies,
glomerulonephritis, generation of Ly6Chi monocytes, and IFN-I production all were greatly attenuated. Lymphocyte activation
following pristane injection was greatly diminished in IRF52/2 mice, and Th cell differentiation was deviated from Th1 in wildtype mice toward Th2 in IRF52/2 mice. Th cell development was skewed similarly in TLR72/2 or IFNAR2/2 mice, suggesting that
IRF5 alters T cell activation and differentiation by affecting cytokine production. Indeed, production of IFN-I, IL-12, and IL-23 in
response to pristane was markedly decreased, whereas IL-4 increased. Unexpectedly, plasmacytoid dendritic cells (pDC) were not
recruited to the site of inflammation in IRF52/2 or MyD882/2 mice, but were recruited normally in IFNAR2/2 and TLR72/2
mice. In striking contrast to wild-type mice, pristane did not stimulate local expression of CCL19 and CCL21 in IRF52/2 mice,
suggesting that IRF5 regulates chemokine-mediated pDC migration independently of its effects on IFN-I. Collectively, these data
indicate that altered production of IFN-I and other cytokines in IRF52/2 mice prevents pristane from inducing lupus pathology by
broadly affecting T and B lymphocyte activation/differentiation. Additionally, we uncovered a new, IFN-I–independent role of
IRF5 in regulating chemokines involved in the homing of pDCs and certain lymphocyte subsets. The Journal of Immunology,
2012, 188: 4113–4121.
4114
Moldawer (Department of Surgery, University of Florida). TLR72/2 mice
on a BALB/c background were acquired from Oriental Bioservices (Kyoto,
Japan), and IFNAR2/2 mice backcrossed nine generations onto a BALB/c
background were provided by Dr. Joan Durbin (Nationwide Children’s
Hospital, Ohio State University, Columbus, OH), respectively. Wild-type
BALB/cJ, B6, and BALB/C 3 B6 F1 CB6F1/J mice were purchased from
The Jackson Laboratory (Bar Harbor, ME). Mice received a single i.p.
injection of 0.5 ml pristane (2,6,10,14-tetramethylpentadecane [TMPD];
Sigma-Aldrich, St. Louis, MO) filtered through a 0.25-mm filter or left
untreated as controls These studies were approved by the Institutional
Animal Care and Use Committee.
Real-time quantitative PCR
Flow cytometry
Flow cytometry was performed as described previously (20). Before surface
staining, peritoneal or peripheral blood cells were incubated with antimouse CD16/32 (Fc Block; BD Biosciences, San Jose, CA) for 10 min.
Cells then were stained with an optimized amount of primary Ab or the
appropriate isotype control for 10 min at room temperature before washing
and resuspending in PBS supplemented with 0.1% BSA. Ten thousand
to 50,000 events per sample were acquired using a CYAN ADP flow
cytometer (Beckman Coulter, Hialeah, FL) and analyzed with FCS Express
3 (De Novo Software). The following conjugated Abs were used: anti–
CD8-allophycocyanin, anti–Ly6G-PE, anti–B220-allophycocyanin–CY7,
anti–CD11c-allophycocyanin, anti–B220-FITC, anti–Ly6C-FITC, CD69FITC, CD69-PE–CY7, CD25-PE (BD Bioscience), anti–H2-Kb–allophycocyanin, anti–H2-Dd–biotin, avidin-PE–CY7, anti–CD11b-Brilliant Violet, anti–pDC Ag-1 (PDCA-1)–biotin (BioLegend, San Diego, CA), anti–
CD4-PE–CY7, and CCR7-PE (eBioscience, San Diego, CA). For morphological analysis, 106 peritoneal cells were cytospun onto glass slides
and stained using the Hema3 kit (modified Wright stain; Fisher Scientific,
Pittsburgh, PA).
Intracellular cytokine staining
Intracellular IFN-g, IL-4, and IL-17 were detected after culturing peritoneal cells or splenocytes at a concentration of 2.5 3 106 cells/ml in RPMI
1640 medium with 10% FBS, PMA (50 ng/ml), ionomycin (1 mg/ml), and
GolgiStop (BD Biosciences; 5 mg/ml) for 6 h at 37˚C in a 5% CO2 incubator. After stimulation, cells were surface stained, fixed in Fixation/
Permeabilization buffer (eBioscience), washed in Perm/Wash buffer
(eBioscience), and then stained with PE-labeled anti–IFN-g, allophycocyanin-labeled anti–IL-4 (BD Biosciences), allophycocyanin-labeled anti–
IL-17 (eBioscience), or isotype controls, according to the manufacturer’s
protocol. Cells were washed twice in Perm/Wash buffer (eBioscience),
resuspended in PBS, and analyzed by flow cytometry. Data were analyzed
with FCS Express 3 software (De Novo Software).
Ig and autoantibody ELISAs
Total Ig levels were measured as described (20). Briefly, microtiter plates
(Immobilizer Amino; Nunc) were coated with goat anti-mouse L-chain (kand l-specific) Abs (Southern Biotechnology Associates, Birmingham,
AL). Sera were diluted 1:200,000 in 150 mM NaCl, 50 mM Tris-HCl (pH
7.5), 2 mM EDTA, 0.5% BSA, 0.3% Nonidet P-40, and 0.05% NaN3
(NET/Nonidet P-40). Alkaline phosphatase-conjugated goat anti-mouse
polyclonal Abs against m, g1, g2c, g2b, or g3 H-chain (1:1000 dilutions; Southern Biotechnology Associates) were used as secondary Abs.
Ag-capture ELISAs for anti-nuclear RNP/Sm Abs were performed as
described (20), using mouse sera at a dilution of 1:400 and goat anti-mouse
IgG second Abs (Southern Biotechnology Associates). Levels of antidsDNA Abs were tested (1:400 serum dilution) by ELISA using S1
nuclease-treated calf thymus DNA as Ag as previously described (22).
Anti-U1A (subset of anti-RNP) Abs were tested by ELISA as described
(23). Briefly, recombinant U1A Ag-coated wells were incubated with
mouse sera (1:400 dilutions) followed by alkaline phosphatase-conjugated
goat anti-mouse m or g1, g2c, g2b, or g3 H-chain-specific Abs. ELISAs
were developed with p-nitrophenyl phosphate substrate (Sigma-Aldrich),
and OD at 405 nm (OD405) was read using a VersaMax microplate reader
(Molecular Devices, Sunnyvale, CA).
Cytokine ELISAs
CCL2 (MCP-1) levels in peritoneal lavage fluid were measured using the
Mouse MCP-1 OptEIA Set (BD Biosciences) following the manufacturer’s
instructions. IL-12 p40/p70 in peritoneal lavage fluid was measured as
described previously (23) using rat mAb pairs for IL-12 (BD Biosciences).
After incubation with biotinylated IL-12–specific Abs, streptavidinconjugated alkaline phosphatase (1:1000 dilution; Southern Biotechnology Associates) was added for 30 min at 22˚C, and the reaction was developed with p-nitrophenyl phosphate substrate. OD405 of each sample was
converted into cytokine concentrations based on the standard curves of
recombinant cytokines using the Softmax Pro 4.3 program (Molecular
Devices) with a four-parameter logistic equation.
Autoantibody analysis by immunoprecipitation
Immunoprecipitation of radiolabeled cell extracts to detect serum autoantibodies against nuclear RNP/Sm was performed as previously described
(20).
Assessment of glomerulonephritis
Glomerular cellularity was evaluated by counting the number of nuclei per
glomerular cross-section after staining with H&E as described (24). For
assessing renal immune complex deposition, 4-mm frozen sections were
stained with FITC rat anti-mouse complement component C3 mAb
(Cedarlane Laboratories) and examined by fluorescence microscopy (24).
Bone marrow chimeras
Bone marrow chimeras were generated as described (25). Briefly, lethally
irradiated (1000 rad) BALB/c 3 B6 F1 (CB6F1/J) mice were reconstituted
with bone marrow from wild-type CB6F1/J mice (H2-Kb– and H2-Dd–
positive) mice mixed 1:1 with bone marrow from either wild-type BALB/c
or BALB/c IFNAR2/2 mice (H2-Dd single positive). After 6 wk, the
reconstituted mice were treated with 0.5 ml pristane i.p., and 3 wk later, the
presence of activated (CD4+CD69+) T cells was determined in the peritoneum and spleen.
Statistical analysis
Statistical analyses were performed using Prism 4.0 software (GraphPad, La
Jolla, CA). Differences between groups were analyzed by the unpaired
Student t test or the Mann–Whitney U test. The data are shown as mean 6
SD for normally distributed data sets and as median and interquartile range
for nonnormally distributed data sets. Student t test was used for normally
distributed data and the Mann–Whitney U test for nonnormally distributed
data. Normality was determined by D’agostino and Person omnibus normality test using Prism 4.0 (GraphPad). All tests were two-sided, and p ,
0.05 was considered significant.
Results
Intraperitoneal pristane injection induces autoantibodies in two
phases: an early phase, peaking at ∼2 wk, of IgM anti-ssDNA and
chromatin autoantibodies and a later phase (3–6 mo) in which
autoantibodies characteristic of SLE are produced, including IgG
anti-Sm/RNP and anti-dsDNA (19). Most of these autoantibodies
are IgG2a in BALB/c background mice (IgG2c in B6 background
mice) or IgG2b. As pristane-induced lupus (autoantibodies and
renal disease) is dependent on TLR7-stimulated IFN-I production
(20), we examined the role of IRF5, a transcription factor down-
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Quantitative PCR (Q-PCR) was performed as previously described (20, 21).
In brief, total RNA was extracted from 106 peritoneal cells using TRIzol
reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized using the
Superscript II First-Strand Synthesis kit (Invitrogen) according to the
manufacturer’s protocol. SYBR Green Q-PCR analysis was performed
using an Opticon II thermocycler (Bio-Rad, Hercules, CA). Amplification
conditions were as follows: 95˚C for 10 min, followed by 45 cycles of 94˚
C for 15 s, 60˚C for 25 s, and 72˚C for 25 s. After the final extension (72˚C
for 10 min), a melting-curve analysis was performed to ensure specificity
of the products. Primer sequences are listed as follows: IFN-stimulated
gene (ISG)-15 forward, 59-GAGCTAGAGCCTGCAGCAAT and reverse,
59-TAAGACCGTCCTGGAGCACT-39; IRF7 forward, 59-ACAGCACAGGGCGTTTTATC-39 and reverse, 59-GAGCCCAGCATTTTCTCTTG-39;
Mx-1 forward, 59-GATCCGACTTCACTTCCAGATGG-39 and reverse,
59-CATCTCAGTGGTAGTCCAACCC-39; CXCL5 forward, 59-CCCCTTCCTCAGTCATAGCC-39 and reverse, 59-TGGATTCCGCTTAGCTTTCT-39; YM-2 forward 59-CTGGGTAATGAGTGGGTTGG-39 and reverse,
59-ACGTCCCTGGTGACAGAAAG-39; YM-1 forward, 59-TGAAGGAGCCACTGAGGTCT-39 and reverse, 59-CACGGCACCTCCTAAATTGT39; Fizz1 forward, 59-TGCTGGGATGACTGCTACTG-39 and reverse, 59AGCTGGGTTCTCCACCTCTT-39; and IL-23 P19 forward, 59-CATGGGGCTATCAGGGAGTA-39 and reverse, 59-GACCCACAAGGACTCAAGGA-39.
PLEIOTROPIC EFFECTS OF IRF5 ON LUPUS PATHOGENESIS
The Journal of Immunology
stream of TLR7 and MyD88, in pristane-induced IFN-I production.
IRF5 is critical for the pathogenesis of pristane-lupus
1C). IgG2c, IgG2b, and IgG1 anti-U1A Abs were absent in
IRF52/2 mice. IgG3 and IgM anti-U1A Abs were not produced by
either IRF52/2 or wild-type mice (data not shown).
Consistent with our previous observation that pristane does not
induce IgG anti-dsDNA Abs in B6 mice (26), little or no antidsDNA Abs were detected in control or in IRF52/2 mice (Fig.
1D). To evaluate whether the absence of autoantibodies in IRF52/2
mice was related to defective isotype switching, we measured total
IgM as well as IgG subclass levels (Fig. 1E). Although pristanetreated IRF52/2 mice had very little serum IgG2c, they produced
significant levels of IgG2b and actually had increased levels of
IgG1. IgM levels were comparable in IRF52/2 and control mice,
as reported previously (6). Nevertheless, IgG1, IgG2b, and IgG3
anti-U1A autoantibodies were not induced in IRF52/2 mice (Fig.
1C and data not shown). The low level of IgG2c (IFN-g dependent) and high level of IgG1 (IL-4 dependent) correlated well with
changes in Th cell subsets (see below).
The relatively mild renal disease induced by pristane in B6 mice
also was attenuated in IRF52/2 mice. H&E staining of the glomeruli revealed decreased cellularity in pristane-treated IRF52/2
mice versus controls as determined by counting the numbers of
nuclei per glomerulus (Fig. 2A, top panel, 2B). Similarly, direct
immunofluorescence staining of glomerular C3 deposition was
diminished in IRF52/2 mice, indicating decreased immune complex deposition in glomeruli (Fig. 2A, bottom panel). Thus,
IRF52/2 mice exhibited substantially decreased autoantibody
production and attenuated renal pathology following pristane
treatment in comparison with wild-type mice.
Decreased inflammatory cytokine production in IRF52/2 mice
Pristane-induced lupus is abolished in the absence of signaling
through the IFNAR (24). Consistent with the importance of IFN-I
in the pathogenesis of this disease, peritoneal exudate cells from
FIGURE 1. Absence of autoantibodies in IRF52/2 mice. IRF52/2 and
control B6 mice (B6 mice, n = 7; IRF52/2 mice, n = 14) were treated with
pristane, and 6 mo later, serum autoantibody and Ig levels were evaluated.
Sera collected from mice before pristane (TMPD) injection served as
a control (Ctr). (A) Anti-Sm/RNP autoantibody levels (ELISA) in IRF52/2
and B6 control mice. (B) Immunoprecipitation of 35S-radiolabeled protein
components A–G of U1 small RNPs by sera from six B6 and six IRF52/2
sera. Positions of molecular mass markers (kDa) are indicated on the right.
(C) Anti-U1A IgG1, IgG2b, and IgG2c autoantibody levels (ELISA) in
IRF52/2 and control mice (B6 mice, n = 7; IRF52/2 mice, n = 14). (D)
IgG anti-dsDNA autoantibody levels (ELISA) in IRF52/2 and control
mice (B6 mice, n = 6; IRF52/2 mice, n = 8). Dotted line indicates the
mean absorbance using sera from non–pristane-treated control mice. (E)
Serum levels of total IgM, IgG1, IgG2c, IgG2b, and IgG3 in pristanetreated IRF52/2 and B6 mice (B6 mice, n = 7; IRF52/2 mice, n = 14).
*p , 0.05, **p , 0.01, ***p , 0.001 by Mann–Whitney U test. ND,
Not detected.
FIGURE 2. Attenuated renal disease in pristane-treated IRF52/2 mice.
(A) Top panel, Kidneys from IRF52/2 mice and wild-type B6 controls 6
mo after pristane treatment were sectioned and stained with H&E. Bottom
panel, Renal tissue was embedded in OCT, and 4-mm sections were stained
with FITC-conjugated anti-mouse complement component C3 mAb.
Original magnification 3200. (B) Glomerular cellularity was evaluated by
counting the number of nuclei per glomerular cross-section (n = 5 mice/
group, eight glomeruli per mouse). ***p , 0.001, Student t test.
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Pristane-treated B6 IRF52/2 mice (n = 14) did not produce antiSm/RNP autoantibodies detectable by ELISA, and only one
mouse was weakly positive by immunoprecipitation (Fig. 1A, 1B).
In contrast, two out of seven wild-type B6 mice treated with
pristane produced high levels of anti-Sm/RNP, and another four
displayed low levels of anti-Sm/RNP detectable by ELISA but not
immunoprecipitation (Fig. 1A, 1B). The frequency of anti-Sm/
RNP in wild-type mice detectable by immunoprecipitation was
consistent with our previous studies in which these autoantibodies
were found in 24% pristane-treated female B6 mice (26). IgG
autoantibodies specific for the U1A protein, a subset of anti-RNP,
were absent in all IRF52/2 mice (n = 14). In contrast, two out of
seven control mice produced high levels and several more produced lower levels of anti-U1A autoantibodies (Fig. 1C). As expected, in wild-type mice, there was a strong IgG2c anti-U1A
response as well as a weaker IgG2b and IgG1 response (Fig.
4115
4116
PLEIOTROPIC EFFECTS OF IRF5 ON LUPUS PATHOGENESIS
IRF52/2 mice expressed markedly lower levels of the ISGs IRF7,
ISG15, and Mx1 (Fig. 3A). Similarly, protein levels of the IFN-I–
inducible chemokine MCP-1 (CCL2) in peritoneal lavage fluid
were significantly lower in IRF52/2 mice (Fig. 3B). The levels of
other proinflammatory cytokines (IL-12 p40/p70 and IL-6) involved in the pathogenesis of pristane-lupus also were decreased
in peritoneal lavage fluid (Fig. 3C) and expression of IL-23 p19
mRNA was decreased in pristane-treated IRF52/2 mice versus
controls (Fig. 3D). Taken together, these data suggest that deficiency of IRF5 attenuates lupus in pristane-treated mice by affecting the production of IFN-I and other key cytokines (IL-12 and
IL-23) that link innate immunity to the activation of Th1 and Th17
cells, respectively (27). Production of IL-4 (Fig. 4) and the chemokine CXCL5, which recruits neutrophils to peritoneal cavity
(28), was similar in IRF52/2 mice and controls, suggesting that
the effects of IRF5 deficiency on cytokine production were selective for Th1 cells and not due to an overall attenuation of the
inflammatory response (Fig. 3D).
We have shown previously that a subset of inflammatory monocytes
expressing CCL2 and high levels of the surface marker Ly6C is
recruited to the peritoneum in response to MCP-1 and is a major
source of IFN-I in pristane-treated mice (29). The total number of
peritoneal cells in pristane-treated IRF52/2 mice was slightly lower
than in wild-type mice but much higher than in untreated mice,
indicating that pristane could induce an inflammatory response in
IRF52/2 mice (Fig. 5A). The effect of IRF5 deficiency on the recruitment of Ly6Chi monocytes (CD11b+Ly6ChiLy6G2) also was
examined (Fig. 5B–E). As shown previously in TLR72/2 and
IFNAR2/2 mice, peritoneal exudates from pristane-treated IRF52/2
mice contained few Ly6Chi monocytes in comparison with
pristane-treated B6 controls (Fig. 5B, R1, 5C, 5D). In contrast, the
peritoneal exudates from IRF52/2 mice contained many Ly6Clo
monocytes (Fig. 5B, R3, 5C, 5D). Peritoneal neutrophils were
largely unaffected (Fig. 5B, R2, 5D). Gating on all monocytes
(CD11b+, Ly6G2) and analyzing fluorescence intensity of Ly6C
FIGURE 4. Inflammatory response to pristane is skewed toward Th2
cells in IRF52/2 mice. Two weeks after pristane injection, peritoneal
lymphocytes were analyzed by flow cytometry. (A) Flow cytometry of
IFN-g+, IL-4+, and IL-17+ cells in total peritoneal exudate cells from
IRF52/2 versus B6 control mice. (B) Quantification of IFN-g–, IL-4–, and
IL-17–producing peritoneal cells. (C) Quantification of CD4+Foxp3+
(regulatory T cell-like) cells in IRF52/2 versus B6 mice. (D) Th1/Th2 ratio
in peritoneal exudates of IRF52/2 versus B6 mice and TLR72/2, IFNAR2/2,
and wild-type BALB/c mice 2 wk after pristane injection. Representative
of two independent experiments; n = 6–14 mice/group. *p , 0.05, **p ,
0.01, ***p , 0.001 by Mann–Whitney U test.
staining underscored the dramatic difference in the profile of
peritoneal monocyte populations in IRF52/2 mice versus controls
(Fig. 5C). In addition, monocytes in the peritoneal exudates had
different morphological features: those in B6 mice had the appearance of immature monocytes, whereas those in IRF52/2 mice
were larger and more granular, consistent with a more mature
phenotype (Fig. 5E). Because Ly6Chi monocytes are a major source
of IFN-I production in the inflamed peritoneum of pristane-treated
mice (29), these results are consistent with the low IFN-I expression
in peritoneal exudates cells from IRF52/2 mice (Fig. 3A). Interestingly, these differences in myeloid cell subsets (Ly6Chi and
Ly6Clo monocytes) were not reflected in the circulation 2 wk following pristane treatment (Supplemental Fig. 1A).
IRF5 deficiency alters the Th cell response to pristane
FIGURE 3. Decreased levels of inflammatory cytokines and chemokines
in IRF52/2 mice. IRF52/2 and B6 mice were injected with pristane, and 2
to 3 wk later, the peritoneum was lavaged, and peritoneal exudate cells
were collected by centrifugation for RNA isolation. The supernatant was
used to assay cytokine/chemokine levels. (A) Expression of the ISGs IRF7,
ISG15, and MX-1 in peritoneal exudate cells (Q-PCR). (B) Level of MCP1 (CCL2) in peritoneal lavage fluid (ELISA). (C) Levels of IL-6 and IL-12
P40/P70 in peritoneal lavage fluid (ELISA). (D) Expression of IL-23a
(p19) and CXCL5 in peritoneal exudate cells (Q-PCR). *p , 0.05, **p ,
0.01, ***p , 0.001 by Mann–Whitney U test.
IRF5 has a critical role in the development of Th1 responses to
Leishmania donovani infection (30). Therefore, we examined the
Th cell response to pristane. There were significant differences in
the Th subsets present in the peritoneum 2 wk after pristane injection. Intracellular cytokine staining revealed that IRF52/2 mice
had fewer total peritoneal exudate cells producing IFN-g, whereas
IL-4– and, to a lesser degree, IL-17–producing cells were increased (Fig. 4A, 4B). Although the IL-4–producing cells in
IRF52/2 mice were primarily T lymphocytes, the cells producing
IL-17 were CD42CD82 (data not shown). To clarify whether the
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IRF5 deficiency alters the chronic inflammatory response to
pristane
The Journal of Immunology
4117
apparent Th cell skewing is due to the lack of IRF5, IFN-I, or
other inflammatory cytokines such as IFN-g and IL-12, we investigated Th cell subsets in IFNAR2/2 and TLR72/2 mice and
found that both IFN-g– and IL-4–producing (but not IL-17) cells
were decreased in these strains (Supplemental Fig. 1B). As in
IRF52/2 mice, the ratio of Th1 to Th2 cells (defined as the ratio of
IFN-g+ to IL4+ T cells) was lower in IFNAR2/2 and TLR72/2
mice versus their wild-type controls (Fig. 4D). It should be noted
that because B6 is a Th1 strain, whereas BALB/c is Th2 prone
(31), the baseline ratios of IFN-g+ to IL-4+ T cells in the two wildtype control strains were quite different (Fig. 4D). Nevertheless,
the cytokine pattern exhibited the same trend in both strains, and
both B6 and BALB/c mice are susceptible to pristane-lupus, although there are differences in autoantibody frequency and the
severity of nephritis (19). Finally, it is of interest that the altered
proportions of Th cell subsets were seen only at the primary site of
inflammation (the peritoneal cavity) and not in the spleen (Supplemental Fig. 1C) and that the percentages of Foxp3+CD4+
(regulatory T cell-like) cells were comparable in IRF52/2 versus
B6 control mice in the peritoneum (Fig. 4C).
The recent observation that IRF5 promotes M1 macrophage
polarization (32), along with the greatly reduced IL-12 (a major
product of M1 macrophages) and IL-23 levels in peritoneal washings
from IRF52/2 mice (Fig. 3) led us to hypothesize that deficiency
of IRF5 may polarize monocyte/macrophage differentiation toward
the M2 pathway in pristane-treated mice. We therefore examined
the expression of Ym1, Ym2, and Fizz1, genes characteristically
expressed by alternatively activated (M2) macrophages (Supplemental Fig. 1D). We did not find a significant difference in the expression
of these genes in peritoneal exudate cells from pristane-treated
IRF52/2 mice versus controls. Taken together, these data suggest
the altered Th cell ratio seen in IRF52/2 mice may reflect decreased
IFN-I production and altered inflammatory cytokine production.
Decreased lymphocyte activation in IRF52/2 mice
The percentages and total cell counts of B220+, CD4+, and CD8+
lymphocytes in peritoneal exudates of pristane-treated mice were
similar in IRF52/2 and wild-type mice (not shown). However, the
percentage of CD4+, CD8+, and B220+ peritoneal exudate cells
expressing the activation marker CD69 was decreased in IRF52/2
mice versus controls (Fig. 6A). Interestingly, the percentage of
activated peritoneal CD69+CD4+ cells was highly correlated with
the percentage of Ly6Chi monocytes (Fig. 6B), suggesting that
IFN-I production, which also correlates strongly with numbers of
Ly6Chi monocytes (29), might promote lymphocyte activation in
pristane-treated mice. Although CD69 is an ISG (33), the presence
of two distinct populations of CD4+ T cells, CD69+ and CD692
(Fig. 6A), argues against the possibility that increased expression
of this surface marker in pristane-treated wild-type mice was
merely a reflection of IFN-I production and suggests that increased CD69 surface staining was due to T cell activation. This
possibility also is supported by the observation that CD69+CD4+
T cells produced substantially more IFN-g and IL-4 than CD692
CD4+ cells in B6 as well as IRF52/2 mice (Fig. 6C). Gated on the
CD4+CD69+ population, ∼30% of B6 T cells produced IFN-g
(Th1) and ∼8% produced IL-4 (Th2) versus ∼20% cells producing
IFN-g and IL-4, respectively, in IRF52/2 mice (Fig. 6C).
To further verify the role of IRF5-stimulated IFN-I in CD4+
T cell activation, we examined bone marrow chimeras. Bone
marrow cells from wild-type BALB/c 3 B6 F1 (CB6F1/J) mice
(bearing both H2-Kb and H2-Dd) were mixed 1:1 with bone
marrow cells from wild-type BALB/c or BALB/c IFNAR2/2 mice
(H2-Dd single positive) and injected into lethally irradiated
CB6F1/J recipients. Six weeks after reconstitution, pristane
was administered to the reconstituted mice, and 3 wk later,
peritoneal lymphocyte activation was examined (Fig. 6D). T cells
derived from the CB6F1/J donor are H2-Kb+, whereas T cells
derived from the BALB/C or IFNAR2/2 donors are H2-Kb2. In
the control group, there is a mixture of CB6F1/J and BALB/c
donor cells, and T cells from both donors became activated
(CD69+) after pristane injection. In mice reconstituted with
CB6F1/J and IFNAR2/2 cells, only the CB6F1/J-derived T cells
were activated, whereas IFNAR2/2-derived T cells remained
CD692 (Fig. 6D, left panel). In contrast to the peritoneum, CD69
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. Effect of IRF5 deficiency on the inflammatory response to pristane. Two weeks after pristane injection, peritoneal cells were collected from
IRF52/2 and wild-type (B6) controls and analyzed by flow cytometry. (A) Total peritoneal exudate cell count. (B) Analysis of total peritoneal exudate cells by
flow cytometry. Percentages of CD11b+Ly6Chi monocytes (R1), CD11b+Ly6Cint neutrophils (R2), and CD11b+Ly6Clo mature monocytes (R3) were assessed
by flow cytometry as described previously (29). (C) Fluorescence intensity of Ly6C on CD11b+Ly6G2 monocytes in the peritoneal exudates was assessed by
flow cytometry. (D) Percentages of Ly6Chi monocytes (CD11b+Ly6ChiLy6G2), Ly6Clo monocytes (CD11b+Ly6CloLy6G2), and neutrophils (CD11b+Ly6Cint
Ly6G+) in the peritoneal exudates. Quantification was based on (C). (E) Morphology of peritoneal exudate monocytes from pristane-treated wild-type (B6)
versus IRF52/2 mice. n = 14–22 per group, representative of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 by Mann–Whitney U test.
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expression on CD4+ peripheral blood and splenic T cells was
comparable in B6 and IRF52/2 mice (Supplemental Fig. 1E).
Together, these results suggest that: 1) CD4+ T activation is both
IFN-I and IRF5 dependent; 2) the T cells must express IFNAR
endogenously; and 3) the exposure of T cells to IFN-I is local
(peritoneal) and not systemic.
pDC recruitment is IRF5 dependent and IFN-I independent
Unexpectedly, 2 wk after pristane treatment, the number of CD11b2
CD11c+B220+PDCA-1+ pDCs in the peritoneal cavity of IRF52/2
mice was strikingly reduced in comparison with wild-type B6 mice
(Fig. 7A). A similar reduction of pDCs in peritoneal exudates was
seen in MyD882/2 mice, but not in IFNAR2/2 or TLR72/2 mice
(Fig. 7B). This discrepancy appeared to be limited to the pDC
population, as myeloid dendritic cells (CD11b+CD11c+CD8a2)
were found at comparable levels in the peritoneal exudates of
IRF52/2 and wild-type mice (Fig. 7C). In contrast to the peritoneal
exudates, the percentage of pDCs in the bone marrow was similar
in IRF52/2 mice and wild-type controls, both before pristane
treatment and 2 wk after treatment (Fig. 7D). CCR7 is expressed
on T cells and dendritic cells, and its ligands, CCL19 and CCL21,
are essential for the homing of pDCs to lymph nodes (34).
Therefore, we examined the expression of CCL19/CCL21 and
found that both were decreased considerably in peritoneal exudate
cells from IRF52/2 versus control mice (Fig. 7E). Thus, defective
pDC migration in IRF52/2 mice is associated with decreased
production of chemokines that mediate pDC homing. In view of
the MyD88 dependence of pDC migration (Fig. 7B) and the ability
of TLR ligands to enhance CCR7 expression (34), we examined
FIGURE 7. Decreased pDCs in the peritoneum of IRF52/2, but not
TLR72/2 or IFNAR2/2, mice. Two weeks after pristane injection, peritoneal exudate and bone marrow pDCs were quantified by flow cytometry.
(A) pDC staining. CD11b2CD11c+B220+PDCA-1+ cells were defined as
pDC; this cell population was also Ly6C+ (not shown). (B) Left panel,
pDCs in peritoneal exudate cells (PEC) from IRF52/2, MyD882/2, and B6
control mice (n = 5–12/group). Right panel, pDCs in TLR72/2, IFNAR2/2,
and BALB/cJ PEC (n = 6/group). (C) Peritoneal myeloid dendritic cells
(CD11b+CD11c+CD82) were quantified by flow cytometry (n = 9 to 10
mice/group). (D) pDCs in bone marrow of control B6 IRF52/2 and B6
mice before pristane injection (Ctr) and 2 wk after pristane injection (n =
3–5/group). (E) Expression of chemokines CCL19 and CCL21a in PEC by
Q-PCR (n = 4–6/group). (F) Expression of CCR7 on bone marrow and
peritoneal pDCs (CD11b2 CD11c+B220+PDCA-1+) was determined by
flow cytometry (n = 4/group). Data are representative of two independent
experiments. *p , 0.05, **p , 0.01, Mann–Whitney U test.
CCR7 expression levels on bone marrow and peritoneal pDCs
from IRF52/2 mice 2 wk after pristane injection. The fluorescence
intensity of B220+CD11b2CD11c+PDCA-1+ pDCs expressing
CCR7 was similar in bone marrow from B6 and IRF52/2 mice
(Fig. 7F). Unexpectedly, the fluorescence intensity of CCR7 was
lower in peritoneal pDCs from B6 mice than in B6 bone marrow
pDCs, whereas IRF52/2 peritoneal pDCs (although few in number) exhibited similar fluorescence intensity to that in IRF52/2
bone marrow pDCs. Although the explanation is unclear, this could
be due to CCL19/CCL21 blocking an epitope of the CCR7 receptor recognized by the mAb in the B6 controls. These data
suggest that MyD88 expression enhances pDC migration to the
peritoneum by increasing IRF5-regulated CCL19/CCL21 and not
by increasing CCR7 expression.
Discussion
Genetic polymorphisms of IRF5 are strongly linked to susceptibility to SLE in humans (9, 10). Recent studies also link IRF5 to
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FIGURE 6. Role of IRF5 in lymphocyte activation following pristane
treatment. Two weeks after pristane injection, peritoneal lymphocytes were
analyzed by flow cytometry. (A) Left panel, Flow cytometry of CD4 and
CD69 (gated on CD11b2 cells). Right panel, Percentage of CD4+, CD8+,
and B220+ cells that were positive for CD69 was determined. (B) Correlation of CD69+ CD4+ T cells with the percentage of Ly6Chi monocytes.
(C) Intracellular staining of IFN-g and IL-4 in B6 and IRF52/2 mice.
Peritoneal lymphocytes were gated on the CD4+ population and then gated
on the CD69+ and CD692 populations, respectively. Percentages of cells
expressing IFN-g and IL-4 are indicated (n = 4/group). (D) Left panel,
Peritoneal cells from bone marrow chimeric mice were gated on the
CD11b2CD4+ population, and CD69 expression was analyzed by flow
cytometry in cells that were H2-Kb+ (CB6F1/J- derived) or H2-Kb2
(BALB/c or BALB/c IFNAR2/2-derived). Right panel, Quantification of
CD69+CD4+ T cells as a percentage of strain-specific total CD4+ T cells
(n = 4 to 5/group). Data are representative of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 by Mann–Whitney U test.
PEC, Peritoneal exudate cells.
PLEIOTROPIC EFFECTS OF IRF5 ON LUPUS PATHOGENESIS
The Journal of Immunology
the pathogenesis of autoantibodies in both human SLE (35) and
pristane-induced lupus (6) and in MRL mice (17). Although it has
been suggested that the primary effect of IRF5 in pristane-lupus
may involve isotype switching of pathogenic autoantibodies (6),
our data strongly suggest that the influence of IRF5 on the development of autoimmune disease in pristane-lupus is multifactorial. In the present studies, IRF5 not only affected autoantibody
production, isotype switching, and the development of renal disease, but also modulated the polarization and activation of Th
cells as well as the differentiation and migration of inflammatory
monocytes/macrophages and pDCs.
Effect of IRF5 deficiency on autoantibody production and renal
disease
IRF5 is critical for inflammatory cytokine production and
recruitment of Ly6Chi monocytes
Intraperitoneal injection of pristane induces the production of inflammatory cytokines, influx of immune cells, and a chronic inflammatory response, culminating in the development of a lupus-like
disease (20). IRF5 deficiency had critical effects on the i.p. production of multiple cytokines implicated in the pathogenesis of
pristane-lupus, including IL-12, IL-23, and IL-6 (Fig. 3). Although
this could be related to overall differences in cytokine production,
it should be pointed out that the gene expression data reflect the
presence of several cell types in the peritoneal exudates, so that
differences in cytokine production also could be related to altered
composition of the peritoneal exudate cells, as exemplified by the
predominance of Ly6Chi monocytes in controls versus Ly6Clo
monocytes in IRF52/2 mice (Fig. 5A). IL-17 production by nonT cells also was affected by the absence of IRF5, but the present
studies did not identify the peritoneal cell subset(s) producing IL17 following pristane treatment. This will require further investigation.
Although the role of IRF5 in the induction of IFN-I is tissue
specific and apparently of minor importance in some cases (1, 36),
IRF5 appears to be essential for the expression of IFN-I–regulated
genes in the pristane model (Fig. 3A). IRF52/2 mice also failed to
produce the IFN-I–inducible chemokine CCL2 (MCP-I, Fig. 3B),
which mediates the recruitment of Ly6Chi monocytes through
CCR2 (21). Similar to the findings in IFNAR2/2 mice, the small
number of monocytes migrating to the peritoneal cavity of
pristane-treated IRF52/2 mice rapidly acquired a Ly6Clo phenotype (Fig. 5) indicative of a more mature phenotype and enhanced
phagocytic capacity. Taken together with our previous findings
(21), IRF5 functions downstream of TLR7, MyD88, IL-1R–associated kinase (IRAK)-4, IRAK-1, and IRAK-2 to promote the
production of IFN-I, MCP-I, and other proinflammatory chemokines and cytokines (21). In contrast, granulocyte migration was
unaffected by the absence of IRF5 (Fig. 5D), suggesting that IRF5
does not participate in the IL-1a–dependent pathway of granulocyte recruitment in this model (28).
IRF5 deficiency alters Th cell activation
TLR7-mediated IRF5 activation is necessary for protective Th1
responses to L. donovani (30). Our data illustrate that TLR7 and
IRF5 also contribute to the activation and polarization of Th1 cells
in pristane-treated mice (Figs. 4, 6). Peritoneal exudates from
wild-type mice contained two distinct subsets of CD4+ T cells:
one CD69+ and the other CD692 (Fig. 4C). The CD69+ subset was
largely absent in IFR52/2 mice. The effect of IRF5 on T cell
activation could be either T cell intrinsic or mediated via its
effects on IFN-I production. The IFNAR dependence in bone
marrow chimera experiments (Fig. 6D) and the correlation between the number of CD69-expressing T cells and type I IFNproducing Ly6Chi monocytes (Fig. 6B) provide evidence that the
effect of IRF5 on T cell activation by pristane is mediated, in part,
by IFNAR signaling. Although CD69 is an IFN-I–responsive gene
(33), the absence of CD69+CD4+ T cells in pristane-treated
IRF52/2 mice may reflect more than just the low levels of IFNI. In pristane-treated B6 mice, one population of CD4+ T cells
expressed CD69, but another subset had no detectable CD69
staining, suggesting that CD69 expression may have significance
beyond being a marker of IFN-I exposure (Fig. 6A). The fact that
intracellular IFN-g staining was preferentially seen in the CD69+
CD4+ subset provides strong evidence that the CD69+ T cells were
not only exposed to IFN-I but also activated (Fig. 6C). Thus, our
data suggest that IRF5-induced IFN-I promotes T cell activation,
consistent with previous observations that IFN-I also promotes the
survival of activated T cells (37).
Pristane-induced lupus is augmented by a Th1 response, as
both autoantibody production and renal disease are attenuated
in pristane-treated IFN-g–deficient mice, whereas disease is exacerbated in IL-42/2 mice (38). The current study shows that in
wild-type mice, the predominance of a Th1 response occurs as
early as 2 wk after pristane injection (Fig. 4). This profile was
limited to the inflamed peritoneal cavity, a site of IFN-I production, and it was not apparent in the spleen (Supplemental Fig. 1).
Interestingly, the Th1/Th2 ratio was strikingly altered in IRF52/2
mice, mainly due to increased numbers of IL-4–producing cells
(Fig. 4). Similar findings were evident in IFNAR2/2 and TLR72/2
mice (Fig. 6, Supplemental Fig. 1), suggesting that the shift toward a Th2 response in IRF52/2 is due to impairment of IFN-I
production rather than an intrinsic effect of IRF5 expression in
T cells.
IRF5 deficiency has an IFN-I–independent effect on pDCs
An unexpected finding of this study was the near absence of pDCs
in the inflamed peritoneal cavity of IRF52/2 or MyD882/2 mice 2
wk after TMPD injection (Fig. 7). In striking contrast, a normal
number of pDCs were present in the peritoneum of pristanetreated TLR72/2 or IFNAR2/2 mice, indicating that the lack of
peritoneal pDCs was not a consequence of low IFN-I levels. The
numbers of pDCs in the bone marrow of IRF52/2 mice were
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IRF5 promotes B cell maturation in part by modulating the expression of Blimp-1, a master regulator of plasma cell maturation
(5). As they age, IRF52/2 mice have more CD19+B2202 plasmablasts and a decreased number of CD138hiB220hi plasma cells
(5). The effects on serum Ig levels are complex: serum levels of
IgG subclasses in young naive IRF52/2 mice are similar to wildtype, whereas in older mice, IgG1, IgG2a/IgG2c, and IgG2b are
decreased. Following immunization with T cell-dependent or
-independent Ags, IRF52/2 mice have a reduced Ag-specific IgG1
response, although IgG2a and IgG2b were not examined (5). In
a previous study on pristane-induced lupus, the production of
IgG2a/IgG2c and IgG2b anti-Sm and anti-dsDNA Abs was abolished in IRF52/2mice (6). Our data are generally consistent with
these observations, although we have not been able to detect antidsDNA Abs in pristane-treated wild-type B6 mice (Fig. 1) (19).
Strikingly, despite increased levels of total IgG1 in IRF52/2 mice,
IgG1 anti-Sm/RNP autoantibodies were not produced, suggesting
that IRF5 may affect the regulation of tolerance to the Sm/RNP
autoantigens.
Although wild-type B6 mice are relatively resistant to the development of glomerulonephritis following pristane treatment
(19), we confirmed that glomerular cellularity and renal immune
complex deposits were decreased in IRF52/2 mice versus wildtype controls.
4119
4120
similar to controls, indicating that IRF5 deficiency does not cause
decreased production of pDCs but instead results in defective
recruitment of these cells to the inflamed peritoneum. The observation that CCL19 and CCL21 expression was abolished in the
peritoneal cavity of IRF52/2 mice following pristane injection
(Fig. 7E) strongly suggests that IRF5 regulates the expression of
these chemokines. In contrast, the expression of CCR7 was similar
or possibly increased in IRF52/2 mice (Fig. 7F), suggesting that
IRF5 affects pDC migration mainly through regulation of CCL19
and CCL21 and not via the CCR7 receptor. The mechanism
for chemokine modulation by IRF5 is unclear. This could be
explained by either direct transcriptional regulation at the promoter region of CCL19/CCL21 or by indirect activation downstream of inflammatory mediators (other than IFN-I) regulated by
IRF5 and MyD88. As CCL19 and CCL21 also play a role in the
recruitment of naive and central memory T cells, future studies to
further characterize the T cell populations found in the peritoneal
exudate and ectopic lymphoid tissue of pristane-treated mice are
warranted.
We thank Dr. Katherine Fitzgerald (University of Massachusetts, Worcester, MA) for providing B6 IRF52/2 mice and Dr. Joan Durbin (Nationwide
Children’s Hospital, Ohio State University, Columbus, OH) for providing
BALB/c IFNAR2/2 mice. We also thank Dr. Eric Sobel (University of
Florida, Gainesville, FL) for advice on the generation of bone marrow
chimeric mice. This paper is dedicated to the memory of Michael J. Keefer, President and Chief Executive Officer of the Lupus Foundation of
Florida.
11.
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21.
Disclosures
The authors have no financial conflicts of interest.
22.
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