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Molecular Immunology 87 (2017) 94–101

Contents lists available at ScienceDirect

Molecular Immunology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m o l i m m

nterleukin-1 receptor associated kinase 1 is a potential therapeutic arget of anti-inflammatory therapy for systemic lupus erythematosus

ingfang Li a, Datang Yu b, Bing Ni c, Fei Hao a,∗

Department of Dermatology, Southwest Hospital, PLA, Third Military Medical University, Chongqing 400038, China Department of Orthopdics, Xinqiao Hospital, PLA, Third Military Medical University, Chongqing 400037, China Institute of Pathophysiology, PLA, Third Military Medical University, Chongqing 400038, China

r t i c l e i n f o

rticle history: eceived 7 February 2017 eceived in revised form 17 March 2017 ccepted 27 March 2017 vailable online 18 April 2017

a b s t r a c t

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease and currently has no effective therapy. The genome-wide analyses indicate that interleukin-1 receptor associated kinase 1 (IRAK1) is associated with the susceptibility of SLE in humans. In the present study, we identified that IRAK1 was overexpressed and hyper-activated in splenic mononuclear cells from B6.MRL-Faslpr/Nju (B6.lpr) mice and peripheral blood mononuclear cells (PBMCs) from SLE patients. Intraperitoneal treat-

eywords: nterleukin-1 receptor associated kinase 1 RAK1 inhibitor uclear factor-kappa B ystemic lupus erythematosus eripheral blood mononuclear cells

ment with a small molecular inhibitor of IRAK1 (IRAK1/4 inhibitor or IRAK-Inh) significantly mitigated inflammatory responses and renal injury in B6.lpr mice. IRAK-Inh treatment or knockdown of IRAK1 by specific siRNA decreased the relative levels of NF-�Bp65 phosphorylation in human PBMCs from SLE patients. Therefore, IRAK1 may be a potential target for anti-inflammatory therapy for SLE and other inflammatory diseases.

© 2017 Elsevier Ltd. All rights reserved.

. Introduction

Systemic lupus erythematosus (SLE) is a representative pro- otype autoimmune disease affecting mainly females (Cooper t al., 2010), and is characterized by chronic inflammation, organ njury and overproduction of various autoantibodies. Current SLE reatment options include immunosuppressive agents and anti- nflammatory drugs. Although tumor necrosis factor � (TNF-�) is an nflammatory factor and a therapeutic target for treatment of joint- elated autoimmune diseases, like rheumatoid arthritis (Murphy t al., 2013), the therapeutic efficacy is limited (Aringer et al., 2012). urrently, the pathogenesis of SLE still remains unclear. It is well nown that the dysregulation of inflammatory signaling during the isease process contributes to the pathogenesis of SLE. Therefore, entral molecules in initiation of an inflammatory cascade may be

potentially effective target for development of therapies for SLE. The IRAK family includes intracellular kinases, which are impor-

ant in the innate immune system. The interleukin-1 receptor ssociated kinase 1 (IRAK1), a serine/threonine kinase, is one of

he IRAK family members and is a key factor in Toll-like (TLR) nd interleukin-1 receptor (IL-1R) signaling (Flannery and Bowie, 010). Engagement of TLR/IL-1R by their ligands induces TLR/IL-

∗ Corresponding author. E-mail address: [email protected] (F. Hao).

ttp://dx.doi.org/10.1016/j.molimm.2017.03.018 161-5890/© 2017 Elsevier Ltd. All rights reserved.

1R activation to recruit myeloid differentiation factor 88 (MyD88), which further recruits IRAK1 and IRAK4 through death-domain interaction. Subsequently, IRAK4 activates IRAK1 by phosphorylat- ing on Thr209 (Cao et al., 1996). The activated IRAK1 interacts with TRAF6 (Kollewe et al., 2004) to facilitate the activation and nuclear translocation of NF-�B, leading to production of pro-inflammatory cytokines, such as IL-1�, IL-6, TNF-� and others (Moynagh, 2009). This pro-inflammatory cascade is crucial for the pathogenesis of SLE. Therefore, inhibition of the IRAK1 function may be valuable in minimizing pro-inflammatory cascade-mediated tissue damages.

Many studies have shown that IRAK1 gene polymorphisms are associated with the susceptibility of SLE in humans (Jacob et al., 2007; Han et al., 2009; Kaufman et al., 2013). How- ever, the exact mechanisms underlying the biological function of IRAK1 have remained unclear. A previous study has reported that IRAK1-deficient mice are resistant to develop SLE-like symptoms, including high levels of serum IgM and IgG autoantibodies, aberrant activation of lymphocytes and dendritic cells, and kidney damage (Jacob et al., 2009). These suggest that IRAK1 is an important deter- minant of the pathogenesis of SLE. Therefore, we hypothesize that inhibition of IRAK1 activity may restrain inflammation in a mouse model of SLE and human SLE patients.

In this study, we examined the relative levels of IRAK1, I�B� and NF-�Bp65 expression and phosphorylation in splenic mononuclear cells from lupus-prone mice and peripheral blood mononuclear cells (PBMCs) from SLE patients. Furthermore, we determined effect

M. Li et al. / Molecular Immuno

Table 1 The demographic and clinical characteristics of SLE patients.

Symptom Number

Total 10 Age (mean year, range) 32.5 ± 18.5 Sex Female Active/inactive disease 7/3 Skin symptoms 8 Anti-dsDNA antibodies 3 Low complement 3 9 Renal disorder 6

o N O i

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Prednisone 10 Immunosuppressive agents 3

f IRAK1 inhibitor (IRAK-Inh) on the relative levels of IRAK1 and F-�B expression and activation as well as kidney tissue damages. ur results reveal that IRAK1 may be a potential target for anti-

nflammatory therapy for SLE and other inflammatory diseases.

. Materials and methods

.1. Subjects and treatment

A total of 10 female patients were randomly selected from our npatient clinic. All patients with SLE were diagnosed, according o the revised SLE criteria of the American College of Rheuma- ology (ACR) (Hochberg, 1997). Their demographic and clinical haracteristics as well as drug treatments were recorded and are hown in Table 1. All of the patients were treated with prednisone 5–30 mg daily) and 3 patients were also treated with methotrexate 10–12.5 mg). Another 8 female and age-matched healthy sub- ects were recruited and served as the normal controls (NC). Their eripheral venous blood samples were obtained. Written informed onsent was obtained from individual subjects and the experi- ental protocol was approved by the Ethics Committees of the

outhwest Hospital.

.2. Mice

Female B6.lpr mice at 12 weeks of age were purchased from

he Model Animal Research Center of Nanjing University, Nanjing, hina. Gender- and age-matched C57BL/6 mice were purchased

rom the Experimental Animal Center of the Third Military Medical niversity and served as the normal controls. All mice were bred

ig. 1. IRAK1 is overexpressed and activated in splenic mononuclear cells from B6.lpr m hat had been treated with vehicle alone or IRAK-Inh and the relative levels of IRAK1 exp nternal control. Data are representative images or expressed as the mean ± SEM of indivi lot analysis; (B) Quantitative analysis of the relative levels of IRAK1 expression and ph roup; ## p < 0.01 vs. the B6.lpr+ vehicle group).

logy 87 (2017) 94–101 95

and housed in a specific pathogen-free facility with free access to autoclaved food and sterile water. The experimental protocol was approved by the Ethics Committee of the Third Military Medical University.

2.3. Cell isolation and culture

B6.lpr and C57BL/6 mice at 14 weeks of age were anesthetized intraperitoneally with sodium pentobarbital (50 mg/kg), and then sacrificed by cervical dislocation. Their splenic mononuclear cells were prepared by Ficoll density gradient centrifugation and total proteins of some splenic mononuclear cells were extracted using the T-PER Tissue Protein Extraction Reagent with 1% protease and phosphatase inhibitors (ThermoFisher, USA). The protein concen- trations of individual samples were quantified with BCA Protein Assay kit (Beyotime Biotechnology, China).

PBMCs were isolated from individual SLE patients and con- trol subjects by Ficoll density gradient centrifugation. The cells at 1 × 106 cells/well were treated in triplicate with, or without, 10 �M IRAK-Inh (Sigma Aldrich, I5409) in 24-well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 �g/ml of streptomycin at 37 ◦C in 5% CO2 for 24 h. Their total proteins were extracted for Immunoblot.

2.4. Treatment with IRAK-Inh in vivo

Female B6.lpr mice at 12 weeks old were injected intraperi- toneally with IRAK-Inh (2.5 mg/kg body weight in DMSO) in 100 �l DMSO three times a week for 2 consecutive weeks. Control C57BL/6 mice were treated with vehicle alone. Two weeks later, their peripheral blood samples were collected for preparing the serum samples. Their splenic mononuclear cells were prepared and their kidney tissues were collected.

2.5. Western blot analysis

Total proteins (20 �g) from each group of splenic mononu- clear cells and human PBMCs were separated by electrophoresis using NuPAGE on 4–12% Bis-Tris Gel (Invitrogen, USA) and trans- ferred onto polyvinylidene fluoride membranes (PVDF, Millipore,

USA), followed by blocking with the Blocking Buffer (ThermoFisher) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 ◦C with anti-phospho-IRAK1 (T209, Assay Biotech, USA), anti-IRAK1, anti-phospho-I�B� (S32 + S36), anti-

ice. Splenic mononuclear cells were isolated from B6.lpr mice and C57BL/6 mice ression and phosphorylation were determined by Western blot using �-actin as an dual groups of mice (n = 3 per group) from three separate experiments. (A) Western osphorylation in splenic mononuclear cells. (** p < 0.01, vs. the C57BL/6+ vehicle

96 M. Li et al. / Molecular Immunology 87 (2017) 94–101

Fig. 2. IRAK-Inh ameliorates lupus-related renal injury in mice. B6.lpr and C57BL/6 mice were treated with vehicle alone or IRAK-Inh (2.5 mg/kg i.p. 3 times/week) for two weeks. Two weeks later, their kidney tissues were subjected to histology by H&E staining and immunofluorescent assays using anti-IgG (red) and anti-C3 (green). The cumulative glomerular activity score (GAS) and tubulointerstitial activity score (TIAS) in individual mice were evaluated in a blinded manner. Data are representative images (magnification, ×1000 for H&E staining, 1200 for immunofluorescent staining) or expressed as the mean ± SEM of individual groups (n = 3 per group) from three separate e immu T + vehi r

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xperiments. (A) Histologic analysis of mouse kidney tissue sections; (B) Anti-IgG IAS. (* p < 0.05; ** p < 0.01 vs. the C57BL/6+ vehicle group; # p < 0.05 vs. the B6.lpr eader is referred to the web version of this article.)

�B�, anti-phospho-NF-�Bp65 (S536), anti-NF-�Bp65 (Abcam, SA), or anti-�-actin (Santa Cruz, USA). After being washed with .05% Tween-20 in TBS, the bound antibodies on the membranes ere detected using an appropriate horseradish peroxidase-

onjugated secondary antibody and visualized using the enhanced hemiluminescent reagents. The relative levels of target proteins o the control �-actin were determined by densitometric analysis sing Image J software.

.6. Immunocytochemistry

The splenic mononuclear cells and PBMCs were smeared on lides, and fixed in 4% paraformaldehyde for 10 min at room tem- erature. After being washed with PBS, the slides were pretreated ith 0.1% of Triton in PBS and blocked with 10% of goat serum in PBS

or 1 h at room temperature. The slides were incubated with rab- it anti-mouse NF-�Bp65 antibody overnight at 4 ◦C. After being ashed, the slides were stained with Alexa Fluor 488-conjugated

oat anti-rabbit IgG (ThermoFisher) and counterstained with DAPI

nofluorescent staining; (C) Anti-C3 immunofluorescent staining; (D) The GAS and cle group). (For interpretation of the references to colour in this figure legend, the

(Beyotime Biotechnology). The fluorescent signals were captured using a laser-scanning confocal microscope (Leica SP5, Germany).

2.7. Pathology examination of the kidney injury

One dissected kidney tissue from individual mice was fixed with 4% paraformaldehyde and paraffin-embedded. The kidney tissue sections (4 �m) were stained with hematoxylin and eosin (H&E) and examined under a light microscope. The severity of renal damages was graded, according to glomerular inflammation, prolif- eration, and crescent formation in individual sections using a score of 0–3. The total scores of individual kidney tissues from three sec- tions selected randomly were calculated, as reported previously (Mishra et al., 2003). The interstitial changes were also recorded.

2.8. Immunofluorescence staining

The remaining kidney of each mouse was prepared for frozen sections (5 �m). After being blocked with 10% of goat blood serum,

M. Li et al. / Molecular Immunology 87 (2017) 94–101 97

Fig. 3. IRAK-Inh inhibits the NF-�B activation in splenic mononuclear cells from B6.lpr mice. B6.lpr and C57BL/6 mice were treated with vehicle or IRAK-Inh for two weeks. Two weeks later, the mice were sacrificed and their spleens were images (A). Their splenic mononuclear cells were isolated and the relative levels of I�B� and NF-�Bp65 expression and phosphorylation to the control �-actin in individual samples were determined by Western blot. Data are representative images or expressed as the mean ± SEM o West v

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f individual groups (n = 3 per group) of mice from three separate experiments. (B) ehicle group; # p < 0.05; ## p < 0.01 vs. the B6.lpr+ vehicle group).

he kidney sections were incubated with rabbit anti-mouse C3 ntibodies (Santa Cruz) at 4 ◦C overnight. Subsequently, the sec- ions were washed with PBS 3 times, and stained with Alexa Fluor 55-conjugated donkey anti-mouse IgG (Beyotime, Biotechnology) nd Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary ntibodies (ThermoFisher). The fluorescent signals were examined nder a fluorescent microscopy.

.9. Enzyme-linked immunosorbent assay (ELISA)

The levels of serum IL-1� and IL-6 in individual mice were deter- ined by ELISA using mouse IL-1� and IL-6 ELISA kits (Dakewe

ioengineering, China), according to the manufacturers’ instruc- ion. The serum samples were diluted at 1:2 and the experimental nd control samples were tested in triplicate simultaneously.

.10. Adenovirus infection

PBMCs from individual SLE patients were infected with Ad- RAK1 shRNA or control Ad-GFP (Hanbio Technology, Shanghai, hina) at a multiplicity of infection (MOI) of 50 for 36 h. he target sequence in IRAK1 for RNA interference is 5′- CAAGTATCTGAAAGACCTGGTGGA-3′.

.11. Statistical analysis

Quantitative data are expressed as mean ± standard error of he mean (SEM). The difference among groups was analyzed by he Mann-Whitney u test or one-way ANOVA using the GraphPad

ern blot analysis; (C) Quantitative analysis. (* p < 0.05; ** p < 0.01 vs. the C57BL/6+

Prism (Version 5.0). A P-value of <0.05 was considered statistically significant.

3. Results

3.1. IRAK1 is overexpressed and activated in splenic mononuclear cells from B6.lpr mice

IRAK1 is a risk factor for SLE (Jacob et al., 2007, 2009). To explore the biological function of IRAK1 in the development of SLE, adult control C57BL/6 and lupus-prone B6.lpr mice were injected with vehicle or IRAK1-Inh for two weeks. Two weeks later, their splenic mononuclear cells were isolated and the relative levels of IRAK1 expression and IRAK1T209 phosphorylation in different groups of cells were determined by Western blot (Fig. 1A). The relative levels of IRAK1 expression in splenic mononuclear cells from lupus-prone B6.lpr mice were significantly higher than that in control mice (p < 0.01, Fig. 1B). Furthermore, treatment with IRAK1-Inh signif- icantly reduced the relative levels of IRAK1 expression in splenic mononuclear cells from both control and B6.lpr mice. A similar pat- tern of IRAK1 phosphorylation was observed in the different groups of cells. Hence, high levels of IRAK1 activation occurred in splenic mononuclear cells of SLE-prone B6.lpr mice.

3.2. Inhibition of IRAK1 activity by IRAK-Inh ameliorates lupus-related renal injury in mice

To determine the effect of IRAK1 inhibition on lupus-related renal injury, B6.lpr and control mice were treated with vehicle or IRAK-Inh for two weeks. The pathological changes in the kid-

98 M. Li et al. / Molecular Immunology 87 (2017) 94–101

Fig. 4. IRAK-Inh inhibits the nuclear translocation of NF-�B in splenic mononuclear cells and reduces pro-inflammatory cytokine production in B6.lpr mice. B6.lpr and C57BL/6 mice were treated with vehicle or IRAK-Inh for two weeks. Two weeks later, their splenic mononuclear cells were isolated and the distribution of NF- �Bp65 expression was determined by immunofluorescent assay. Furthermore, the levels of serum IL-6 and IL-1� in individual mice were determined by ELISA. Data are r 8 per m e hori v

n i C r B l t I fl o d ( i t t a t i s w c m n

epresentative images or expressed as the mean values of individual groups (n = 7– agnification, ×1200. (B) The levels of serum IL-6; (C) the levels of serum IL-1�. Th

ehicle group; # p < 0.05; ## p < 0.01 vs. the B6.lpr+ vehicle group.

eys of different groups of mice were evaluated by histology and mmunofluorescent assays using anti-mouse IgG and anti-mouse 3. While healthy kidney tissues were observed in the control mice, egardless of IRAK-Inh treatment, the kidney tissue sections from 6.lpr mice exhibited typical features of nephritis, such as glomeru-

ar hypercellularity, inflammatory cell infiltration and membrane hickness, which were obviously mitigated in that from the IRAK- nh-treated B6.lpr mice (Fig. 2A). Similarly, while little anti-IgG uorescent signal was detected in the control mice regardless f IRAK-Inh treatment, obvious anti-IgG fluorescent signals were etected in the kidney tissues from the vehicle-treated B6.lpr mice Fig. 2B). The anti-IgG fluorescent signals were remarkably reduced n the kidneys from the IRAK-Inh-treated B6.lpr mice. Moreover, here was little anti-C3 fluorescent signal in the kidney tissues from he control mice and IRAK-Inh-treated B6.lpr mice, but a trance of nti-C3 fluorescent signals was detected in the kidney tissues from he vehicle-treated B6.lpr mice (Fig. 2C). Semi-quantitative analysis ndicated that the cumulative glomerular activity and tubulointer- titial scores in the kidneys from the vehicle-treated B6.lpr mice ere significantly higher than that in the controls and signifi-

antly reduced in the kidneys from the IRAK-Inh-treated B6.lpr ice (Fig. 2D). Thus, inhibition of IRAK1 activity by IRAK-Inh sig-

ificantly mitigated lupus-related renal injury in mice.

group) from three separate experiments. (A) Immunofluorescent analysis. Original zontal bars represent mean ± SEM of individual groups. ** p < 0.01 vs. the C57BL/6+

3.3. Inhibition of IRAK1 activity inhibits the NF-�B activation in splenic mononuclear cells from B6.lpr mice

The aberrant NF-�B activation is associated with the pathogene- sis of autoimmune and inflammatory diseases, such as rheumatoid arthritis and SLE (Okamoto, 2006; Simmonds and Foxwell, 2008). In addition, activation of IRAK1 can activate downstream several signal pathways, including the NF-�B activation (Flannery and Bowie, 2010). To understand the consequence of IRAK-Inh treat- ment, B6.lpr and control mice were treated with vehicle or IRAK-Inh for two weeks. The splenic sizes of each group of mice were images and measured in Fig. 3A. The sizes of spleen tissues from B6.lpr mice that had been treated with vehicle alone were obviously larger than that of the IRAK-Inh-treated mice and control mice. Furthermore, the relative levels of I�B� and NF-�Bp65 expression and phospho- rylation in splenic mononuclear cells were determined by Western blot. As shown in Fig. 3B–C, treatment with IRAK-Inh significantly increased the relative levels of I�B� expression, but decreased the relative levels of I�B� phosphorylation in splenic mononuclear cells from both B6.lpr and control C57BL/6 mice, particularly in the

cells from B6.lpr mice. As a result, the relative levels of NF-�Bp65 in the splenic mononuclear cells from the vehicle-treated B6.lpr mice were significantly higher than that in the control mice and

M. Li et al. / Molecular Immunology 87 (2017) 94–101 99

Fig. 5. IRAK-Inh attenuates the IRAK1-NF-�B signaling in PBMCs from SLE patients. PBMCs were obtained from eight SLE patients or NC. Two to three PBMC samples from patients or NC were pooled and treated in triplicate with, or without, IRAK-Inh for 24 h. The relative levels of IRAK1, I�B� and NF-�Bp65 expression and phosphorylation in individual subjects were determined by Western blot and the nuclear translocation of NF-kB was determined by immunofluorescent assay. Data are representative images or expressed as the mean ± SEM of individual groups (n = 8 per group) from three separate experiments. (A) Dose-dependent inhibition of IRAK1 expression. (B) Western blot analysis. (C) Quantitative analysis. (D) immunofluorescent analysis (Original magnification, ×1200.). ** p < 0.01 vs. the NC+ vehicle group; ## p < 0.01 vs. the patients+ vehicle group.

100 M. Li et al. / Molecular Immunology 87 (2017) 94–101

Fig. 6. Inhibition of IRAK1 by IRAK1-shRNA attenuates the NF-kB signaling in PBMCs from SLE patients. PBMCs from three SLE patients were isolated and infected with Ad-IRAK1-shRNA or control Ad-shRNA for 36 h. Some PBMCs were treated with IRAK-Inh or vehicle for 24 h. The relative levels of IRAK1, I�B� and NF-kBp65 expression a ages s .05; ** g

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nd phosphorylation were determined by Western blot. Data are representative im eparate experiments. (A) Western blot analysis; (B) Quantitative analysis. (* p < 0 roup).

ignificantly reduced in the IRAK-Inh-treated B6.lpr mice. A simi- ar pattern of the relative levels of NF-�Bp65 phosphorylation was etected in the different groups of cells. Further immunofluores- ent analysis indicated that treatment with IRAK-Inh dramatically educed the nuclear translocation of NF-�Bp65 in both B6.lpr and ontrol mice, as compared with the vehicle-treated mice (Fig. 4A). LISA revealed that higher levels of serum IL-6 and IL-1� were etected in the vehicle-treated B6.lpr mice, as compared with hat in the control mice. The levels of serum IL-6 and IL-1� were ignificantly reduced in the IRAK-Inh-treated B6.lpr mice. Collec- ively, these data indicated that inhibition of IRAK1 by the IRAK-Inh

itigated the I�B� phosphorylation and degradation, leading to nhibition of the NF-�B activation in splenic mononuclear cells and ystemic IL-6 and IL-1� responses in B6.lpr mice.

.4. High IRAK1 activation leads to aberrant activation of the F-�B signaling in PBMCs from SLE patients

Next, we determined the effect of IRAK-Inh on the levels f IRAK1 and NF-�B activation in PBMCs from 8 patients with LE and 8 healthy controls by Western blot and immunoflu- rescent assays. First, we found that treatment with IRAK-Inh own-regulated IRAK1 expression and phosphorylation in a dose- ependent manner (Fig. 5A). Furthermore, the relative levels of

RAK1 and NF-�Bp65 expression and phosphorylation in PBMCs rom SLE patients were significantly higher than that from healthy ontrols. Treatment with IRAK-Inh dramatically reduced the rela- ive levels of IRAK1 and NF-�Bp65 expression and phosphorylation n PBMCs from SLE patients (Fig. 5B and C). In comparison with he healthy controls, significantly lower levels of I�B� expression, ut higher levels of I�B� phosphorylation were detected in PBMCs

rom SLE patients. Moreover, treatment with IRAK-Inh obviously revented the nuclear translocation of NF-�B in PBMCs from SLE atients (Fig. 5D). Collectively, high levels of IRAK1 activation led berrant activation of the NF-�B signaling in PBMCs, which was

or expressed as the mean ± SEM of individual groups (n = 3 per group) from three p < 0.01 vs. the NC+ vehicle group; # p < 0.05; ## p < 0.01 vs. the patients+ vehicle

mitigated by treatment with IRAK-Inh. Thus, inhibition of IRAK activity by IRAK-Inh mitigated I�B� phosphorylation and reduced the NF-�B activation in PBMCs.

3.5. Knockdown of IRAK1 expression mimics the effects of IRAK-Inh on inhibiting the NF-kB signaling in PBMCs from SLE patients

To validate the effects of the IRAK-Inh, some PBMCs from 3 indi- vidual SLE patients were infected with Ad-shRNA or Ad-GFP for 36 h, respectively. Some PBMCs were treated with vehicle alone or IRAK-Inh for 24 h. Subsequently, the relative levels of IRAK1, I�B�, NF-�Bp65 expression and phosphorylation in individual groups of cells were determined by Western blot. In comparison with that in the vehicle-treated control cells, treatment with IRAK-Inh significantly reduced the relative levels of IRAK1, NF-�Bp65 expres- sion and phosphorylation as well as I�B� phosphorylation, but increased the levels of I�B� expression in PBMCs (Fig. 6). A similar pattern of IRAK1, I�B�, NF-�Bp65 expression and phosphorylation was detected in individual groups of Ad-GFP or Ad-shRNA-infected cells, demonstrating that knockdown of IRAK1 mimicked the effect of IRAK-Inh treatment in PBMCs from SLE patients.

4. Discussion

Effective therapy of SLE is challenging because treatment- related side effects commonly occur in most SLE patients. Therefore, it is essential to identify new molecular targets for devel- opment of new therapeutic reagents to control SLE progression. In this study, our results suggest that IRAK1 may be a new target for intervention of SLE. First, we found significantly upregulated levels

of IRAK1 expression and NF-�B activation in splenic mononuclear cells from lupus-prone B6.lpr mice and PBMCs from SLE patients. These data support the notion that IRAK1 is a risk factor for devel- opment of SLE (Zhai et al., 2013) and aberrant activation of the

muno

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1682–1689. Zhai, Y., Xu, K., Leng, R.X., et al., 2013. Association of interleukin-1

receptor-associated kinase (IRAK1) genepolymorphisms (rs3027898, rs1059702) with systemic lupus erythematosus in aChinese Han population. Inflamm. Res. 62 (6), 555–560.

M. Li et al. / Molecular Im

F-�B signaling contributes to the pathogenesis of SLE in humans Wong et al., 1999; Pacheco et al., 2016). Furthermore, inhibition f IRAK1 expression by miRNAs promotes DC apoptosis, suggest- ng that IRAK1 positively regulates the survival of DC and enhances nflammation (Park et al., 2015). Thus, IRAK1 may contribute to the athogenesis of SLE and other inflammatory diseases by enhancing he NF-�B signaling, pro-inflammatory cytokine production and DC urvival.

Previous studies have shown that mangiferin can ameliorate olitis by inhibiting the IRAK1 phosphorylation and the NF-�B sig- aling (Jeong et al., 2014). Furthermore, inhibition of IRAK1/4 by a mall molecule or specific shRNA significantly reduces T-ALL cell roliferation (Li et al., 2015). In this study, we found that treatment ith IRAK-Inh significantly attenuated the IRAK1 expression and F-�B activation in splenic mononuclear cells from lupus-prone 6.lpr mice and PBMCs from SLE patients. Treatment with IRAK-Inh ignificantly mitigated lupus-related renal damage by significantly educing the glomerular activity score (GAS) and tubulointersti- ial activity score (TIAS) as well as IgG and C3 kidney deposition in 6.lpr mice. Treatment with IRAK-Inh significantly inhibited I�B� nd NF-�Bp65 phosphorylation and NF-�Bp65 nuclear transloca- ion in splenic mononuclear cells from B6.lpr mice, accompanied by ignificantly reducing serum levels of pro-inflammatory IL-6 and IL- �. Furthermore, treatment with IRAK-Inh significantly attenuated

RAK1 expression, the NF-�B activation and nuclear translocation n human PBMCs from SLE patients. Similarly, IRAK1 silencing by RAK-specific shRNA not only decreased the IRAK1 expression, but lso inhibited the NF-�B activation in human PBMCs from SLE atients. These novel data suggest that inhibition of IRAK1 may itigate I�B� phosphorylation, which in turn inhibits the NF-�B

ignaling and downstream pro-inflammatory cytokine production n immunocompetent cells, leading to alleviation of lupus-related idney injury. Indeed, recent genome-wide association studies GWAS) of SLE in diverse ancestral populations have identified that RAK1 is a risk factor for development of SLE (Alarcon-Riquelme t al., 2016). IRAK1-deficient mice are resistant to SLE induction Jacob et al., 2009). Furthermore, IRAK1 is associated with aber- ant activation of the NF-�B signaling in the pathogenic process of nflammatory diseases (Liu et al., 2007). Hence, inhibition of IRAK1 ctivity may be a valuable strategy to develop new therapies for LE and other inflammatory diseases.

In summary, our data indicated higher levels of IRAK1 xpression and activation in splenic mononuclear cells from upus-prone B6.lpr mice and human PBMCs from SLE patients. nhibition of IRAK1 by IRAK-Inh attenuated the NF-�B signal- ng and lupus-related renal damages, accompanied by reducing ro-inflammatory cytokine production in B6.lpr mice. Similarly, reatment with IRAK-Inh or IRAK1-specific shRNA was down- egulated the NF-�B signaling in human PBMCs from SLE patients. hese suggest that IRAK1 may be an important factor, contributing o the pathogenesis and a new therapeutic target for interven- ion of SLE and other pro-inflammatory diseases. We recognized hat our study had limitations, including small sample size, lack f dose–dependent curves of IRAK-Inh in functional study and a ingle time point. We are interested in further determining the herapeutic applicability of IRAK1 inhibitors.

ompeting interests

The authors declared no conflicts of interest in this work.

inancial support

This work was supported by a grant from the National Natural cience Foundation of China (No. 81472883).

logy 87 (2017) 94–101 101

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