Glucocorticoids suppress inflammation via the

Full text

Turn on search term navigation

ARTICLE

Received 23 Oct 2014 | Accepted 9 Dec 2014 | Published 14 Jan 2015

Masanori Miyata1,*, Ji-Yun Lee1,*, Seiko Susuki-Miyata1, Wenzhuo Y. Wang1,2, Haidong Xu1, Hirofumi Kai3, Koichi S. Kobayashi4, Richard A. Flavell5 & Jian-Dong Li1

Glucocorticoids are among the most commonly used anti-inammatory agents. Despite the enormous efforts in elucidating the glucocorticoid-mediated anti-inammatory actions, how glucocorticoids tightly control overactive inammatory response is not fully understood. Here we show that glucocorticoids suppress bacteria-induced inammation by enhancing IRAK-M, a central negative regulator of Toll-like receptor signalling. The ability of glucocorticoids to suppress pulmonary inammation induced by non-typeable Haemophilus inuenzae is signicantly attenuated in IRAK-M-decient mice. Glucocorticoids improve the survival rate after a lethal non-typeable Haemophilus inuenzae infection in wild-type mice, but not in IRAK-M-decient mice. Moreover, we show that glucocorticoids and non-typeable Haemophilus inuenzae synergistically upregulate IRAK-M expression via mutually and synergistically enhancing p65 and glucocorticoid receptor binding to the IRAK-M promoter. Together, our studies unveil a mechanism by which glucocorticoids tightly control the inammatory response and host defense via the induction of IRAK-M and may lead to further development of anti-inammatory therapeutic strategies.

1 Center for Inammation, Immunity & Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia 30302, USA. 2 Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA. 3 Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan. 4 Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, Texas 77843, USA. 5 Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.-D.L. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

DOI: 10.1038/ncomms7062 OPEN

Glucocorticoids suppress inammation via the upregulation of negative regulator IRAK-M

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062

Haemophilus inuenzae (NTHi), not only in airway epithelial cells, but also in macrophages. We found that the overexpression of IRAK-M suppresses, whereas IRAK-M depletion enhances, the NTHi-induced expression of proinammatory mediators. We further found that IRAK-M-deciency attenuates the ability of dexamethasone (DEX) to suppress pulmonary inammation induced by NTHi infection. Lethal NTHi infection-caused mortality was improved by DEX treatment in wild type (WT), but not in IRAK-M-decient mice. Together our results suggest that the induction of IRAK-M by GCs may be critical to suppress overactive pulmonary inammatory response in vitro and in vivo.These results thus identied IRAK-M as a novel functional target of GCs.

ResultsGCs synergistically enhance NTHi-induced IRAK-M expression.We sought to determine if GCs regulate the expression of IRAK-M induced by NTHi. Because airway epithelial cells are the front line of defense through initiating inammatory response, we rst examined the effect of GCs on NTHi-induced expression in human respiratory epithelial cells BEAS-2B and A549. DEX synergistically enhanced the NTHi-induced IRAK-M expression at the mRNA level in BEAS-2B and A549 in a dose- and time-dependent manner (Fig. 1a,b and Supplementary Fig. 1a,b).Immunoblot analysis revealed that DEX also synergistically enhanced NTHi-induced IRAK-M expression at the protein level (Fig. 1c,d). Moreover, DEX-mediated synergistic enhancement of NTHi-induced IRAK-M expression is also conrmed in primary normal human bronchial epithelial cells (Fig. 1e). We also examined the effect of GCs on IRAK-M expression in macrophages due to their important role in innate immune responses against bacteria23,24. As shown in Fig. 1f, the induction of IRAK-M by NTHi and DEX was also observed in primary human monocyte-derived macrophages. Under the same experimental condition, NTHi-induced IL-6 expression was suppressed by DEX (Supplementary Fig. 2). Consistent with these results, DEX also synergistically enhanced the NTHi-induced IRAK-M expression in primary alveolar macrophages of mice (Fig. 1g).Moreover, DEX also synergistically enhanced the NTHi-induced IRAK-M expression at both mRNA and protein levels in lung tissue of mice, as assessed by performing qPCR and immunostaining analyses (Fig. 1hj). To further determine which cells express IRAK-M in the lung of mice treated with NTHi and DEX, we next performed immunouorescence double staining using anti-IRAK-M with anti-E-cadherin or anti-F4/80 antibodies, the marker of epithelial cells and macrophages, respectively. As shown in Fig. 1k, NTHi and DEX markedly induced the expression of IRAK-M in both epithelial cells and macrophages. Of note, IRAK-M-specic staining was completely abolished by an IRAK-M-specic blocking peptide (Fig. 1k).

Figure 1 | DEX synergistically enhances the NTHi-induced IRAK-M expression in vitro and in vivo. (a) IRAK-M mRNA expression in human bronchial epithelial BEAS-2B cells treated with DEX (0.01, 0.1, 1, 10, 100 or 1,000 nM) for 1 h, followed by the stimulation of NTHi for 5 h. (b) IRAK-M mRNA expression in human lung epithelial A549 cells stimulated with DEX (100 nM) for 1 h, followed by NTHi for indicated time. (c) A549 cells were treated with DEX (100 nM) for 1 h, followed by NTHi for 12 h. IRAK-M protein was detected by anti-IRAK-M antibody. (d) IRAK-M protein expression was quantied from three independent experiments. (e) IRAK-M mRNA expression in primary NHBE cells treated with DEX (10, 100 or 1,000 nM) for 1 h and subjected to NTHi stimulation for 5 h. (f) Human peripheral blood CD14 monocytes were differentiated to macrophages by culturing them with granulocyte

macrophage colony-stimulating factor for 7 days. IRAK-M mRNA was determined after the macrophages were treated with DEX (100 nM) for 1 h, followed by stimulation of NTHi for 5 h. (g) Mice were stimulated with DEX (1 mg kg 1, intraperitoneally) for 2 h and intratracheally inoculated with NTHi(1 107 c.f.u.) for 24 h. The mRNA expression in alveolar macrophages was assessed by qPCR. (h) The IRAK-M mRNA expression in lung was assessed by

qPCR. (i) Immunostaining of mouse lung with control IgG or anti-IRAK-M antibody by LSAB (Labelled Streptavidin Biotin) staining system (Scale bar, 50 mm; magnication, 400). (j) Treatment-blind observers scored the IRAK-M expression from the histology results. (k) The lung sections were stained

by using indicated antibodies. The arrows and arrowheads indicate the epithelial cells and macrophages merged with IRAK-M expression, respectively. (Scale bar, 50 mm; magnication, 400. Data in a,b,d-h, n 3; j, n 6) are means.d. *Po0.05; t-test.

Glucocorticoids (GCs) are the most widely used and most effective treatment to control inammatory diseases13. GCs are known to exert their anti-inammatory effects

by binding to glucocorticoid receptors (GRs), leading to the suppression of proinammatory regulators such as nuclear factor-kB (NF-kB) or activator protein 1 (AP-1) (refs 4,5).

Several mechanisms of action of GR have been reported in the past6. First, GR binds to p65 and AP-1 to prevent downstream transcription (tethering). Second, GR binds to the glucocorticoid response element (GRE) to initiate the transcription of anti-inammatory genes (transactivation). Third, negative GRE has been shown to suppress proinammatory cytokines (transrepression)7,8.

The innate immune and inammatory response is activated by pattern recognition receptors, including Toll-like receptors (TLRs), on recognition of pathogen-associated molecular patterns9. Pattern recognition receptors activate a number of downstream molecules such as tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), NF-kB essential modulator, inhibitor of NF-kB (IkB) kinase b (IKKb) and NF-kB to produce proinammatory cytokines9. Myeloid differentiation factor 88 (MyD88) is a critical downstream adaptor molecule of all TLRs, except TLR3, and interleukin-1 (IL-1) receptor (IL-1R) family (IL-1a, IL-1b, IL-18 and IL-33) signalling by recruiting IL-1R-associated kinase 1 (IRAK1), IRAK4 and TRAF6 (refs 9,10). The studies of MyD88-decient mice and humans suggest that MyD88 plays a pivotal role in initiating inammatory responses11,12.

Negative feedback regulators of inammation have been recently suggested to play essential roles in tightly controlling inammatory responses to preserve homeostasis10. IRAK-M (also known as IRAK3) is one of the most critical negative feedback regulators of the TLR/IL-1R family signalling via the inhibition of MyD88 and IRAK1/4 activation1316. IRAK-M is a member of the IRAK family and is composed of three conserved domains. However, it does not have any kinase activity due to the lack of a key aspartate in its kinase domain. Thus, IRAK-M has been thought to be a competitor for IRAK1 in associating with MyD88 and TRAF6 (ref. 13). Indeed, IRAK-M-decient mice exhibited increased inammatory response in several models1519. IRAK-M expression was initially characterized in monocytes/ macrophages16,20,21. Recent studies also demonstrate the expression of IRAK-M in airway epithelial cells20,22. Induction of IRAK-M expression by a number of inammatory stimuli has been shown to suppress inammation in a negative feedback manner in multiple cell types including macrophages and epithelial cells18,19. However, it is unclear if GCs suppress overactive inammatory responses via induction of negative feedback regulators such as IRAK-M.

In the present study, we show that GCs synergistically enhances IRAK-M expression induced by non-typeable

2 NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062 ARTICLE

Taken together, our data suggest that DEX synergistically enhances NTHi-induced IRAK-M expression at both mRNA and protein levels in vitro and in vivo.

IRAK-M negatively regulates the NTHi-induced inammation. To determine the role of the induction of IRAK-M expression in

the NTHi-induced inammatory responses, we established stable BEAS-2B cells expressing IRAK-M (IRAK-M-stable cells; Fig. 2a). As shown in Fig. 2b, the NTHi-induced expression of TNF-a,

IL-1b, IL-6, CXCL10 and CCL5 was signicantly inhibited in IRAK-M-stable cells as compared with mock cells. Next we sought to conrm the role of endogenous IRAK-M expression in

b c d

NTHiNTHi + DEX

DEX

NTHiNTHi + DEX

DEX

Relative quantity of

IRAK-M mRNA

25 CON

NTHi

20 15 10

5 0

Relative quantity of

IRAK-M mRNA

0 0 6 12 18 24Time (h)

100

NTHi

DEX IRAK-M

-actin

+ kDa

Relative intensity of

IRAK-M protein

0 0 3 6

Time (h)

9 12

DEX -

- DEX

Primary NHBE Mouse alveolar

macrophages

Mouse lung

* *

f g h

Primary human

macrophages

10 CON

* *

Relative quantity of

IRAK-M mRNA

Relative quantity of

IRAK-M mRNA

Relative quantity of

IRAK-M mRNA

Relative quantity of

IRAK-M mRNA

NTHi

0 0 0

DEX

NTHi

+ DEX

NTHi +

+ DEX

NTHi +

Mock

DEX

IRAK-M

lgG IRAK-M

lgG

CON NTHi

IRAK-M expression in lung

(Blinded scoring)

NTHi +

DEX

Mock

DEX

CON

NTHi NTHi

E-cad /

IRAK-M/

DAPI

F4/80 /

IRAK-M/

DAPI

IRAK-M

blocking

peptide

IRAK-M/

DAPI

NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062

a b

TNF-

IL-1

IL-6

CXCL10

CCL5

IRAK-M

200

150

100

CON

* * * *

250 200

200

NTHi

Relative quantity

of mRNA

150

100 100

50 50

0 0 0

IRAK-M

IRAK-M (SC)

IRAK-M (PS)

150

2,000

1,500

1,000

500

kDa

-actin

Mock

IRAK-M

+ + + +

c d

IRAK-M

siRNA

* * * * *

Relative quantity

of mRNA

8,000

6,000

4,000

2,000

Mock

140

120

100

120,000

80,000

6,000

4,000

2,000

NTHi

DEX

+ + + +

+ kDa

40,000

0 0 0 0

IRAK-M

siRNA

+ + + + +

TNF- CXCL10 CCL5

Mock

IRAK-M siRNA

1.2 1.2 1.21.0 1.0 1.00.8 0.8 0.80.6 0.6 0.60.4 0.4 0.40.2 0.2 0.20.0 0.0 0.0 NTHi

DEX (nM) 0 0 0

1 1 1

10 10 10

* * *

Relative quantity

of mRNA

+ + + + + + + + +

Figure 2 | IRAK-M suppresses the NTHi-induced proinammatory mediator expression. (a) The proteins from BEAS-2B cells stably overexpressing IRAK-M and its control pcDNA3.1 (Mock) cells were detected by immunoblot with indicated antibodies. (b) The mRNA expression was detected by qPCR after the stable cells were treated with DEX (100 nM) and NTHi. (c) BEAS-2B cells were transfected with siRNA control or IRAK-M for 72 h. IRAK-M protein expression was detected by immunoblot with anti-IRAK-M antibodies (SC, Santa Cruz Biotechnology sc-100389; PS, ProSci #2355).(d) siRNA-transfected BEAS-2B cells were infected with NTHi followed by qPCR. (e) The mRNA level was calculated by following: (NTHi-induced mRNA at each concentration of DEX)/(NTHi-induced mRNA without DEX). Data in b,d,e, n 3) are means.d. *Po0.05; t-test.

NTHi-induced expression of proinammatory mediators by depleting IRAK-M using short interfering RNA (siRNA). Immunoblot analysis using two different anti-IRAK-M antibodies revealed that the siRNA depletion of IRAK-M specically reduced IRAK-M expression (Fig. 2c). IRAK-M depletion markedly enhanced NTHi-induced expression of TNF-a, IL-1b, IL-6,

CXCL10 and CCL5 (Fig. 2d). Moreover, the ability of DEX to suppress these proinammatory mediators was also signicantly attenuated in IRAK-M-depleted cells (Fig. 2e). These data thus provide direct evidence for the role of the induction of IRAK-M expression in NTHi-induced inammatory responses in the presence of DEX.

GCs suppress inammation and improve survival via IRAK-M. To determine if DEX suppresses NTHi-induced inammatory response via the induction of IRAK-M expression, we compared the inhibitory effect of DEX on NTHi-induced innate inammatory response in WT (Irak-m / ) with IRAK-M-decient (Irak-m / ) mice. We observed that DEX markedly suppressed the NTHi-induced mRNA expression of key proinammatory mediators including TNF-a, IL-1b, IL-6, MIP2, CXCL5/LIX,

CXCL10 and CCL5, at the mRNA level, in Irak-m / but not in Irak-m / mice (Fig. 3a). Enzyme-linked immunosorbent assay analysis conrmed that DEX failed to markedly suppress the

NTHi-induced expression of these proinammatory mediators at the protein level in the bronchoalveolar lavage (BAL) uid of

Irak-m / mice as compared with Irak-m / mice (Fig. 3b). Consistent with these results, similar results were also observed in mouse alveolar macrophages (Fig. 3c). Moreover, histopathological analysis of the lung of NTHi-infected mice showed that DEX inhibited NTHi-induced leukocyte inltration in the peribroncheal and interstitial area and also inhibited alveolar wall injury by the inammatory process in Irak-m / but not in

Irak-m / mice (Fig. 4a,b). Similarly, DEX also suppressed NTHi-induced leukocytes inltration in BAL uid of Irak-m / mice but not of Irak-m / mice (Fig. 4c). Because an innate inammatory response initiated by pulmonary epithelial cells is critical for bacterial clearance, we further evaluated the effects of DEX on bacterial clearance in the lungs of Irak-m / mice compared with Irak-m / mice. DEX treatment increased the colony-formation unit (c.f.u.) of NTHi in the lung of Irak-m / mice, but not in the lung of Irak-m / mice (Fig. 4d).

Consistent with the result of bacterial clearance, DEX also inhibited the NTHi-induced expression of mouse b-defensin 4, an orthologue of human BD2, in Irak-m / mice, but not in

Irak-m / mice (Fig. 4e). Because overactive inammatory responses contribute signicantly to the increased morbidity and mortality in patients with NTHi infections25,26, we next evaluated the contribution of IRAK-M in mortality induced by NTHi by infecting mice with a lethal dose of NTHi (5 108 c.f.u.) and

monitoring survival rate for 7 days. As shown in Fig. 4f, we observed B50% mortality in Irak-m / mice 7 days after infection. We found no statistically signicant difference in

4 NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062 ARTICLE

Mouse lung CON NTHiTNF- IL-1 IL-6

DEX + +

WT KO

Irak-m WT KO

0 + +

+ +

WT KO WT KO WT KO WT KO WT KO

MIP2 CXCL5/LIX CXCL10 CCL5

Bronchoalveolar lavage (BAL)

TNF- IL-1 IL-6 MIP2 CXCL5/LIX CXCL10 CCL5

* * * *

NS NS

* * *

* NS

Relative quantity

of mRNA

50 40 30 20 10

100 80 60 40 20

120 100

80 60 40 20

7 6

3 2 1 0

CON NTHi

NS NS NS NS NS NS

Protein (pg ml1)

Relative quantity

of mRNA

400

300

200

100

300

200

100

* *

2,000

1,500

1,000

500

600 500

400 300 200 100

500 400 300 200 100

2,000 1,500

1,000 500

1,000 800 600 400 200

DEX + +

+ +

WT KO

Irak-m WT KO WT KO WT KO WT KO WT KO WT KO

Mouse alveolar macrophages

TNF- IL-1 IL-6 MIP2 CXCL10 CCL5

CON NTHi

* NS

200

150

120 100

80 60 40 20

* NS

50 40 30 20 10

60 50 40 30 20 10

100 * *

DEX + +

+ +

WT KO

Irak-m WT KO WT KO WT KO WT KO WT KO

Figure 3 | DEX suppresses the NTHi-induced proinammatory mediator expression in Irak-m / but not in Irak-m / mice. WT and IRAK-M-decient mice were inoculated with DEX (1 mg kg 1, intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 107 c.f.u. per lung) for 24 h.

(a) The mRNA expression in the mouse lung tissue was analysed by qPCR. (b) The protein level of proinammatory mediators in BAL uid was determined by enzyme-linked immunosorbent assay. (c) The mRNA expression in alveolar macrophages was analysed by qPCR. Data in a,c, n 3; b, n 9 are

means.d. *Po0.05, NS, non-signicant; t-test.

survival rate between DEX-untreated Irak-m / and Irak-m / mice (P 0.362). Notably, the survival rate was signicantly

improved by DEX in Irak-m / but not in Irak-m / mice (Fig. 4f). Collectively, our data suggest that DEX suppressed bacteria-induced innate inammatory response and improved survival via the upregulation of IRAK-M expression.

IKKb and GR mediate synergistic induction of IRAK-M. Having shown that GCs suppress NTHi-induced inammatory response via the induction of IRAK-M, the mechanism underlying the regulation of IRAK-M expression still remains unknown. Because NTHi is recognized by TLR2 (refs 2729), we sought to rst evaluate the generalizability of our ndings by determining if DEX also enhances the induction of IRAK-M expression by TLR-dependent and -independent inammatory stimuli. GCs enhanced upregulation of IRAK-M by TNFa, IL-1b and Pam3CSK4 in lung epithelial cells (Fig. 5a). Because all of these stimuli that induce IRAK-M, including NTHi, are known to induce inammatory response via IKKb2729, we thus examined

if DEX synergistically enhances upregulation of IRAK-M induced by activating IKKb signalling through the overexpression of a constitutively active form of IKKb (IKKb CA). DEX indeed synergistically enhanced the induction of IRAK-M by the direct activation of IKKb signalling (Fig. 5b). We further determined if IKKb mediates the synergistic upregulation of IRAK-M by DEX and NTHi. Inhibition of IKKb using specic inhibitor signicantly suppressed the synergistic upregulation of IRAK-M expression at both mRNA and protein levels by DEX and NTHi (Fig. 5c,d), suggesting the requirement of IKKb signalling in mediating the synergistic induction of IRAK-M.

Because GR is crucial for GC-mediated biological effects5,6, we next assessed the role of GR using RU486 (mifepristone), a GR antagonist. RU486 signicantly inhibited the synergistic upregulation of IRAK-M expression at both mRNA and protein levels by DEX and NTHi in BEAS-2B and A549 cells (Fig. 5e,f and Supplementary Fig. 3a). Moreover, GR depletion using siRNA also markedly inhibited the synergistic upregulation of IRAK-M by DEX and NTHi (Fig. 5g and Supplementary Fig. 3b), indicating that GR mediates synergistic induction of IRAK-M.

NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062

Irak-m WT

Irak-m KO

CON

NTHi

CON

NTHi

Mock DEX Inflammation score

b c d

CON

NTHi

4 8

* * *

PMN cell number

in BALF (104 l1)

NTHi in lung

(104c.f.u. g1)

NTHi

DEX

+ + + + + + + +

DEX NTHi

DEX

Irak-m

e f

100

mBD4 CON

NTHi

Relative quantity

of mRNA

0 0 1 2 3 4 5 6 7

% Survival

WT + CONWT + DEX Irak-m KO + CON Irak-m KO + DEX

DEX

WT Days after NTHi infection

Figure 4 | DEX suppresses NTHi-induced innate inammatory response in Irak-m / but not in Irak-m / mice. (ae) WT and IRAK-M-decient mice were inoculated with DEX (1 mg kg 1, intraperitoneally) for 2 h, followed by intratracheal inoculation with NTHi (1 107 c.f.u. per lung) for 24 h.

(a) Haematoxylin and eosin (H&E) staining of lung tissues from mice (Scale bar, 200 mm, magnication, 100 in large frame; Scale bar 50 mm,

magnication, 400 in inserted frame). (b) Blinded histopathologic scoring of lung inammation was performed on H&E-stained lung sections in a

grade 03. (c) The number of PMN cells from BAL uid was counted using a haemocytometer under the microscope. (d) Bacterial loads (c.f.u.) in lung homogenates. (e) The mRNA expression of mBD4 in lung was measured by qPCR (f) WT and IRAK-M-decient mice were inoculated with DEX(1 mg kg 1, intraperitoneally) for 2 h, followed by intratracheal inoculation with lethal dose of NTHi (5 108 c.f.u. per lung) for 7 days. Survival rate

was monitored for indicated days. P values were determined by KaplanMeier survival analysis with GraphPad Prism 5.0. Data in b 8; c,e, n 3; d 10,

f 20 are means.d. *Po0.05, NS, non-signicant; t-test.

GR is known to regulate gene expression by inuencing transcription or mRNA stability30,31. Interestingly, the mRNA stability of IRAK-M remained largely unchanged by NTHi or DEX (Supplementary Fig. 4). Consistent with this result, actinomycin D, a transcriptional inhibitor, abrogated the synergistic induction of IRAK-M protein expression induced by DEX and NTHi (Fig. 5h). These data suggest that DEX and NTHi are unable to induce IRAK-M expression in the absence of the on-going transcription.

We sought to further elucidate the molecular mechanism underlying the synergistic induction of IRAK-M by DEX and NTHi. DEX is known to exert its transcriptional activity via the induction of the ligand-dependent dimerization of GR and the

subsequent binding of the dimerized GR to GRE in the gene regulatory region of the driven genes46. We thus rst determined if the ligand-induced binding of GR dimer to the GRE in the promoter region of IRAK-M is involved in the synergistic induction of IRAK-M transcription by using a non-steroidal GR-monomer-favouring compound, compound A (CpdA). Unlike DEX, CpdA exhibits no effect on GRE-driven gene transcription32. We found that CpdA failed to enhance NTHi-induced expression of IRAK-M (Fig. 5i). In contrast, it still signicantly inhibited the NTHi-induced NF-kB activity (Fig. 5j).

These data thus suggest that the binding of dimerized GR to GRE is required for the synergistic induction of IRAK-M transcription by DEX and NTHi.

6 NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062 ARTICLE

CON

NTHi TNF- IL-1 Pam

50 40 30 20 10

DEX

+ + + +

Relative quantity of

IRAK-M mRNA

kDa

IRAK-M

-actin

IKK CA

DEX

CON

Mock

IKK inh

15 12

Relative quantity of

IRAK-M mRNA

NTHi

+ +

DEX

+ +

kDa

9 6 3

IRAK-M

-actin

DEX

Mock

IKK inh

Mock

RU486

+ +

Relative quantity of

IRAK-M mRNA

+ +

NTHi

DEX

RU486

g h

Mock Mock

GR siRNA

ActD

NTHi NTHi

+ +

DEX DEX

kDa kDa

IRAK-M

60 60

100

18 15 12

9 6 3 0

-actin

** *

* *

i j

NF-B luc

CON CON

Relative quantity of

IRAK-M mRNA

NTHi NTHi

RLA

120 100

80 60 40 20

DEX DEX

CpdA (M) CpdA (M)

1 1

10 10

Figure 5 | DEX synergistically enhances the NTHi-induced IRAK-M expression via IKKb and GR. (a) Immunoblot shows that IRAK-M protein expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, TNF-a (10 ng ml 1), IL-1b (1 ng ml 1) or Pam3CSK4 (1 mg ml 1). (b) IRAK-M mRNA expression assessed by qPCR in A549 cells transfected with constitutive active form of IKKb (IKKb CA) with DEX (100 nM). (cf), IRAK-M expression in BEAS-2B cells treated with DEX (100 nM) and NTHi, IKKb inhibitor (1 mM) (c,d) or RU486 (1 mM; e,f). (g) Immunoblot shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA GR. (h) BEAS-2B cells were treated with NTHi, DEX (100 nM) and actinomycin D, ActD (5 mg ml 1), followed by immunoblot.

(i) The expression of IRAK-M mRNA in BEAS-2B cells treated with DEX (100 nM) and Compound A, CpdA (1 or 10 mM) followed by NTHi stimulation for 5 h. (j) Relative luciferase activity (RLA) of NF-kB is measured by luciferase assay. BEAS-2B cells transfected with NF-kB luciferase plasmid were treated with CpdA and NTHi. Data (b,c,e,i,j, n 3) are means.d. *Po0.05, NS; t-test.

GR and p65 synergistically bind to IRAK-M promoter. We next sought to dene the cis-acting DNA elements critical for the synergistic induction of IRAK-M. We constructed a series of deletion mutants in the upstream region of IRAK-M promoter,

inserted them to a pGL3 basic vector and measured their luciferase activity. As shown in Fig. 6a, the key cis-acting elements critical for mediating the synergistic induction of IRAK-M transcription resided within the promoter region from 500 to 71

NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062

a b c

IRAK-M promoter

300 +71

IRAK-M promoter

RLA

0 LUC GRE

CON

NTHi

DEX

0 2 4 6 8

B #1 B #2, 3

+ 71

LUC

1.9 kb

1.5 kb

1.0 kb

0.5 kb

0 kb

NTHi

LUC

LUC CON DEX

NTHi

CON

RLA

+ +

DEX +

+ +

WT 3 B

GRE

p65 p65+/+

MEF

p65/ MEF

e f

Chlp : GR

300 250 200 150 100

50 0

Chlp : p65

NTHi

DEX

* *

DEX

+ +

kDa

IRAK-M

p65

Mock p65 siRNA

Fold enrichment

500

400

300

200

100

-actin

NTHi

+ + DEX

NTHi

DEX

0 + +

g h i

350 300 250 200 150 100

50 0

1stIP

2ndIP

p65

lgG

250

500 250

Fold enrichment

500

ReChlP Input

NTHi

+ +

++ DEX

NTHi +

+ +

+ + + +

+ + + + +

NTHi

0 +

+ +

DEX

CON RU486

+ +

IKK inh CON RU486

IKK inh

Figure 6 | DEX and NTHi synergistically induce IRAK-M transcription via inducing the binding of p65 and GR to IRAK-M promoter. (ac) Relative luciferase activity (RLA) was measured after the transfection of IRAK-M promoter in pGL3 basic vector and treated with DEX (100 nM) and NTHi in BEAS-2B cells (a,b). (c) MEF cells were transfected with IRAK-M-luc ( 500 bp), followed by DEX (1 mM) and NTHi stimulation. (d) Immunoblot

shows IRAK-M protein expression in BEAS-2B cells transfected with siRNA p65. (e,f) ChIP assay in BEAS-2B cells treated with DEX and NTHi for 1 h. IRAK-M promoter regions from 38 to 39 bp for GRE, from 231 to 99 bp for kB site were amplied by qPCR. (g) Re-ChIP assay (detailed in

Methods). PCR products were detected in agarose gel. (h,i) ChIP assay in BEAS-2B cells treated with IKKb inhibitor (1 mM), RU486 (1 mM), DEX (100 nM) and NTHi. Data (ac, n 3; e,f, n 2) are means.d. *Po0.05; t-test.

base pair (bp). Further in silico sequence analysis revealed the existence of three putative NF-kB-binding sites (kB site) and one

GRE in the IRAK-M promoter region from 300 to 71 bp

(Supplementary Fig. 5). Interestingly, mutation of this GRE and these three kB sites markedly inhibited the synergistic activation of IRAK-M promoter by DEX and NTHi (Fig. 6b), thereby further demonstrating the requirement of NF-kB and GR. Since p65 is a key subunit of NF-kB33, we next determined if p65 is critically involved in this synergistic induction of IRAK-M. We observed no synergistic induction of IRAK-M in p65-decient mouse embryonic broblasts (MEFs) treated with NTHi and DEX as compared with WT MEFs, and the reconstitution of p65-decient MEFs with WT p65 plasmid restored their responsiveness to NTHi and DEX (Fig. 6c). Consistent with these results, p65 knockdown by siRNA inhibited the synergistic induction of IRAK-M expression at the protein level in BEAS-2B

cells (Fig. 6d and Supplementary Fig. 6a) and DEX also synergistically enhanced IRAK-M expression induced by expressing WT p65 in BEAS-2B cells (Supplementary Fig. 6b). Taken together, our data suggest that both p65 and GR are required for mediating the synergistic induction of IRAK-M transcription by DEX and NTHi.

We next determined if DEX synergizes with NTHi to induce IRAK-M transcription via synergistically inducing the binding of p65 and GR to IRAK-M promoter region ( 300 bp to 71 bp)

containing three kB sites and GRE. Quantitative analyses using chromatin immunoprecipitation (ChIP) assays revealed that DEX and NTHi synergistically enhanced the binding of both p65 and GR to IRAK-M promoter (Fig. 6e,f). In addition, Re-ChIP assay34 revealed that DEX and NTHi induced the interaction of p65 with GR in the context of chromatin (Fig. 6g). Moreover, the synergistic induction of the binding of p65 and GR to IRAK-M

8 NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062 ARTICLE

NTHi GCs

Cytoplasm

IRAK IRAK-M

IKK

p65

Nucleus

Inflammation

p50 p50

p65 p65

Cytokines

Chemokines

p50

GR GR GRE

IRAK-M

Figure 7 | A schematic representation of suppression of inammation by GCs via the upregulation of IRAK-M expression.

promoter induced by NTHi and DEX was signicantly inhibited by RU486 and IKKb inhibitor, respectively (Fig. 6h,i). Together these data suggest that DEX synergizes with NTHi to induce the binding of both GR and p65 to IRAK-M promoter, which, in turn, leads to the induction of IRAK-M transcription in a synergistic manner (Fig. 7).

DiscussionOn infection, an innate inammatory response can be activated through a TLR. The adaptor proteins IRAK1 and IRAK4 (IRAK1/4) and MyD88 mediate all TLR signalling, except TLR3, in triggering inammation. It has been demonstrated that MyD88 and IRAK1/4 are critical in activating an inammatory response in mice12,35 as well as in humans11,36. Since GCs have a potent anti-inammatory effect for multiple usages in a clinical setting, we hypothesized that GCs could target central bottleneck proteins such as MyD88 or IRAK1/4 to tightly regulate inammatory responses. Here we show that GCs synergistically enhance bacteria-induced IRAK-M, a critical negative regulator of IRAK1/4, in vitro and in vivo. We found that NTHi and GCs cooperatively induced recruitment of p65 and GR to the IRAK-M promoter. In addition, the inhibitory effect of DEX on NTHi-induced inammatory response is signicantly attenuated in mice lacking IRAK-M. Thus, we propose that IRAK-M is a novel functional target of GCs for suppression of bacteria-induced inammation.

In the present study, we provided experimental evidence for a novel anti-inammatory mechanisms by which GCs suppress inammation via the cooperative upregulation of IRAK-M by the GR and NF-kB. This nding may be of particular signicance as it contradicts a widespread assumption that GCs exert their anti-inammatory effects principally by antagonizing NF-kB activity4,5. If extended to other models of inammatory diseases, our study may have signicant translational implications for design of novel anti-inammatory agents. Previous studies have

predominantly focused on understanding the GC-mediated anti-inammatory actions through the inhibition of positive regulators such as NF-kB and AP-1. Although it has been shown that GCs suppress inammation by targeting more distal regulators such as IkBa or MKP-1 (refs 3739), the role of GCs in regulating more proximal and central regulators of inammation, such as the key bottleneck proteins MyD88 and IRAK1/4, remains unknown. Recently, systematic approaches revealed that MyD88 is a non-redundant core element40,41. Owing to GCs prominence among anti-inammatory agents, it is logical that GCs may target MyD88-IRAK1/4 to tightly control inammation. Thus, our study provides novel insight into the tight regulation of MyD88 and IRAK1/4 via modulating the endogenous inhibitor IRAK-M by GCs.

GR has been shown to suppress inammatory responses via the inhibition of p65-induced transcription4,37,42. A recent genome-wide study revealed that GR and p65 mutually enhance their binding to a specic promoter43. Thus, the regulation of p65- and GR-associated transcription is likely gene specic. Despite previous studies showing that the expression of IRAK-M is induced by inammatory stimuli in macrophages22,4447 and in osteoblasts48, the precise molecular mechanism of IRAK-M regulation at the transcriptional level remains to be understood. We demonstrated that the co-treatment of cells with both NTHi and GCs leads to the synergistic binding of p65 and GR to the IRAK-M promoter. Interestingly, NTHi- and GR-induced binding of p65 to the IRAK-M promoter is inhibited by the GR antagonist, RU486 (Fig. 6h). Conversely, we also observed a decrease in GR binding in the presence of IKKb inhibitor (Fig. 6i). Consistent with previous studies, our data suggest that both p65 and GR synergize with each other to bind to the IRAK-M promoter. These data suggest a novel regulatory mechanism for IRAK-M promoter activity. Future studies investigating how the p65-GR complex activates IRAK-M transcription may lead to a better understanding of the elaborate interplay between bacteria and GCs.

Initially, IRAK-M expression was predominantly characterized in monocytes/macrophages16,20,21. Recently it has been shown that during respiratory infections, IRAK-M is expressed and induced, not only in alveolar macrophages, but also in respiratory epithelial cells22,49,50. In the present study, we found that GCs synergize with NTHi to induce IRAK-M expression in both respiratory epithelial cells and alveolar macrophages in vitro and in vivo. Importantly, GCs are unable to suppress the NTHi-induced upregulation of proinammatory mediators in IRAK-M-depleted lung epithelial cells as well as IRAK-M-decient alveolar macrophages. Our ndings are in line with previous studies demonstrating the expression of IRAK-M in non-myeloid cells including epithelial cells. Thus, it is likely that IRAK-M expressed in both respiratory epithelial cells and macrophages are involved in mediating the ability of GCs to suppress inammation induced by the respiratory bacterial pathogen, NTHi. It should be noted that our study did not exclude the involvement of other cell types. Future studies using conditional knockout mice with tissue-/cell type-specic deletion of IRAK-M may help determine the contribution of cell-specic induction of IRAK-M expression.

IRAK-M has been shown to act as a critical negative regulator for inammatory responses. In line with previous ndings in pneumonia models1520,51, we observed elevated inammatory responses in the lung of IRAK-M-decient mice compared with WT mice. We found that GCs suppressed the NTHi-induced inammation and signicantly improved the survival rate in WT mice but not in IRAK-M-decient mice (Fig. 4f). Interestingly, no statistically signicant difference in survival rate was found between DEX-untreated WT and IRAK-M-decient mice although IRAK-M-decient mice show a trend of better

NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 9

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062

survival. Our nding is consistent with a recent study showing a reduced lethality in IRAK-M-decient mice after infection withS. pneumonia via the airway20. Given that the markers of inammation are indeed elevated in IRAK-M-decient mice, it is unclear why IRAK-M-decient mice did not show an increased mortality. Although the current study does not provide a clear explanation for this apparent anomaly, our preliminary nding on the negative role of IRAK-M in bacteria-induced mucus production may provide possible explanations for this difference. Recently, there is an increasing evidence suggesting that the host has the ability to reduce the tissue damage caused directly by both pathogens and immunopathology through the enhancement of tolerance mechanisms, thus improving host survival52. IRAK-M may act as a negative regulator for NTHi-induced host tolerance. Indeed, our preliminary studies revealed that IRAK-M is also a negative regulator for bacteria-induced mucus production, an important mucosal defense mechanism for protecting the host from inammation- and pathogen-induced tissue damage. Thus, IRAK-M-deciency results in, not only overactive inammation, but also overproduced mucus, which are apparently counteractive. This disparity may explain why IRAK-M-decient mice did not show an increased mortality. Future studies using mice decient in mucus production may help to further address this question.

In conclusion, our studies unveil a novel mechanism by which GCs suppress bacteria-induced innate immune and inammatory responses by upregulating IRAK-M. Our study provides new insights into the previously unidentied role of GCs in suppressing inammation by targeting the central bottleneck proteins MyD88 and IRAK1/4. It may also lead to the development of new therapeutic strategies to control overactive inammation.

Methods

Reagents and antibodies. DEX, Mifepristone (RU486) and Actinomycin D were purchased from Sigma-Aldrich. Compound A was purchased from Enzo Life Science. IKK2 inhibitor IV was purchased from EMD Millipore. Antibodies:GR (sc-8992), p65 (sc-8008, sc-372, sc-109), IRAK-M (sc-100389), a-Tubulin (sc-69969), b-actin (sc-8432), F4/80 (sc-26642), E-cadherin (sc-31020), Donkey anti-rabbit IgG-FITC (sc-2090) and bovine anti-goat IgG-TR (sc-2786) were purchased from Santa Cruz Biotechnology, IRAK-M (#2355) from ProSci, anti-rabbit HRP-linked antibody (#7074) and anti-mouse HRP-linked antibody (#7076) were from Cell Signaling. siRNAs; GR (cat# L-003424-00-0005, ON-TARGET plus Human NR3C1 (2908)-SMARTpool, p65 (cat# L-003533-00-0005, ON-TARGET plus Human RELA (5970) - SMARTpool) and control siRNA (cat# D-001810-10-05) were from Thermo Scientic Dharmacon, IRAK-M (IRAK3 (ID 11213) cat# SR307690 and control siRNA (cat# SR30004) were from OriGene.

Mice and animal experiments. Irak-m / mice have been described previously16, and age-matched (89 weeks old) male C57BL/6 J mice were used as

WT controls. For investigation of the NTHi-induced inammation in mice, anaesthetized mice were intratracheally inoculated with NTHi at a concentration from 1 107 to 5 108 c.f.u. per mouse and saline was inoculated as control. The

inoculated mice were then killed after NTHi inoculation. For PMN analysis, BAL uid was collected by cannulating the trachea with sterilized PBS in mice followed by staining with Diff-Quik staining system (modied Giemsa staining). For isolation of macrophages, we collected BAL uid with 3 ml sterilized PBS.

After centrifuge the BAL uid, macrophages were puried by percoll gradient preparation (Amersham Pharmacia Inc.). For inhibition study, mice were pretreated with DEX (1 mg kg 1) intraperitoneally 2 h before NTHi inoculation.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Georgia State University.

Histology and immunostaining. For histological analysis, formalin-xed parafn-embedded lung tissues were sectioned (4 mm) and then stained with haematoxylin and eosin to visualize inammatory responses and pathological changes in the lung. The stained sections were then imaged and recorded under light systems (AxioVert 40 CFL, AxioCam MRC, and AxioVision LE Image system, Carl Zeiss). The detection of IRAK-M protein was performed using rabbit anti-IRAK-M (ProSci, 2 mg ml 1 for Immunohistochemistry, 10 mg ml 1 for Immunouorescence) and Donkey anti-rabbit-FITC (Santa Cruz Biotechnology,5 mg ml 1) in the parafn section of mouse lung tissue. Blocking IRAK-M peptide

(ProSci, cat# 2355 P, 25 mg ml 1) was used for the negative control experiments. Epithelial cells and macrophages were recognized by antibodies of anti-E-cadherin (Santa Cruz Biotechnology, 10 mg ml 1) and anti-F4/80 (Santa Cruz Biotechnology, 10 mg ml 1), respectively and followed by bovine anti-goat TR (Santa Cruz

Biotechnology, 8 mg ml 1) incubation. Inammation score in haematoxylin and eosin staining (Grade; 0 to 3) and IRAK-M protein expression intensity score in immunostaining (Grade; 0 to 4) were validated in a blinded fashion5356.

Bacterial culture. NTHi strain 12 (also known as R2846) used in this study was a clinically isolated strain that was kindly provided by H. Faden (Childrens Hospital of Buffalo, State University of New York, Buffalo, NY)57. NTHi were grown on chocolate agar plate at 37 C in an atmosphere of 5% CO2 overnight and inoculated in brain heart infusion broth supplemented with 3.5 mg ml 1 NAD and haemoglobin (BD Biosciences). After overnight incubation, bacteria were subcultured into fresh brain heart infusion and the log phase NTHi, monitored by measurement of optical density (OD600) value, was washed and suspended in

DMEM for in vitro cell experiments and in isotonic saline for in vivo animal experiments.

Cell culture. All media described below were supplemented with 10% fetal bovine serum (Sigma-Aldrich). Human airway epithelial A549 cells were maintained in F-12 K media (Gibco), BEAS-2B cells in RPMI 1640 media (Gibco). BEAS-2B cells stably expressing human IRAK-M were obtained by plasmid transfection following geneticin selection (300 mg ml 1). Human primary bronchial epithelial (Lonza)

cells were maintained in BEGM (bronchial epithelial growth media) supplemented with BEGM SingleQuots. Human peripheral blood CD14 monocytes (Lonza)

were cultured in RPMI 1640 media (Gibco) containing 1 mM pyruvate and GM-CSF (50 ng ml 1; R&D systems). MEF cells were obtained from E13 embryos and maintained in DMEM (Corning Cellgro). p65 / MEFs reconstitutedwith p65 WT were cultured in DMEM (Corning Cellgro) containing puromycin(1.5 mg ml 1). All cells were cultured in a humidied atmosphere of 5% CO2 at 37 C.

Real-time quantitative RTPCR analysis. Total RNA was isolated with TRIzol reagent (Life Technologies) by following the manufacturers instruction. The reverse transcription reaction was performed by using 1 mg of RNA in 25 ml of reaction buffer of TaqMan reverse transcription reagents (Applied Biosystems) and run under the following protocol: 25 C for 10 min, 42 C for 1 h and 95 C for5 min (refs 29,5860). PCR was performed by using Fast SYBR Green Master Mix (Life Technologies). In brief, the reactions were performed in triplicate containing 2 Universal Master Mix, 1 ml of template cDNA, 400 nM primers in a nal

volume of 12.5 ml and they were analysed in a 96-well optical reaction plate (Applied Biosystems). Reactions were amplied under the following protocol:95 C for 20 s followed by 40 cycle of 95 C for 3 s and 60 C for 30 s and quantied by using StepOnePlus Real-Time PCR System and the manufacturers corresponding software (StepOnePlus Software v2.3; Applied Biosystems). The relative quantities of mRNAs were obtained by using the comparative Ct method and were normalized using human cyclophilin or mouse glyceraldehydes-3-phosphate dehydrogenase as an endogenous control. The primers are described in Supplementary Table 1.

Plasmids and transfections. The expression plasmid of a constitutively active form of IKKb (IKKb-CA, S177E/S181E) was a gift from Dr Anjana Rao (Addgene plasmid # 11105) (ref. 61). The expression plasmid of p65 was cloned and the insert was transferred to pcDNA3.1 vector (Life Technologies) with BamHI and HindIII sites after we amplied the insert with primers shown in Supplementary Table 2. The luciferase reporter construct of NF-kB contains three copies of the NF-kB site from the IL-2 receptor (a) promoter by using the following oligonucleotides: 50-TCGAGACGGCAGGGGAATCTCCCTCTCCG-30 and 30-CTGCCGTCCCCTTAGAGGGAGAGGCAGCT-50 (refs 27,62). Transient transfections were carried out using TransIT-LT1 reagent (Mirus) or Lipofectamine 2000 (Life Technologies) for plasmid DNA, DharmaFECT4 (Thermo Scientic) for siRNA following the manufacturers instruction. For cloning of human IRAK-M, we used pDONR223-IRAK3 as a template, which was a gift from Drs William Hahn and David Root (Addgene plasmid # 23627)(ref. 63). The insert was then transferred to pcDNA3.1 vector (Life Technologies) with XhoI and BamHI site after we amplied the insert with primers shown in Supplementary Table 2. For cloning of IRAK-M promoter, we used BAC clone RP11-937C6 (BACPAC Resources Center, Childrens Hospital Oakland Research Institute) as a template. PCR was performed using primers shown in Supplementary Table 2. PCR products were transferred into pGL3 basic vector (Promega) with MluI and XhoI site. The sequences were veried from at least three clones. The GRE and NF-kB mutants of IRAK-M promoter were constructed by using the QuikChange II site-directed mutagenesis kit (Agilent Technologies) with primers shown in Supplementary Table 2.

Western blot analysis. Western blots were performed using whole-cell extracts in protein lysis buffer (20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 50 mM Na4P2O7,

10 NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062 ARTICLE

30 mM NaF, 5 mM ZnCl2, 2 mM Iodoacetic acid, 1% Triton-X) with freshly added 1 mM sodium orthovanadate and protease inhibitor cocktail (Sigma-Aldrich), separated on 8% SDSpolyacrylamide gel electrophoresis gels and transferred to polyvinylidene diuoride membranes. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T). The membrane was then incubated in a 1:2,000 dilution of a primary antibody in 5% bovine serum albuminTBS-T at room temperature for 1 h or at 4 C for 16 h. After washing three times with TBS-T, the membrane was incubated with 1:10,000 dilution of the corresponding secondary antibody in 2.5% non-fat dry milkTBS-T at room temperature for 2 h. Respective proteins were developed by using Amersham ECL Prime Regent (GE Healthcare Biosciences) and image were obtained by ChemiDoc XRS System. Images have been cropped for presentation.

Full-size images are presented in Supplementary Figs 79.

Chromatin immunoprecipitation. ChIP assay was performed with minor modications of the previous study42,64. The cells were crosslinked by incubation with 1% formaldehyde at room temperature for 10 min, followed by incubation with0.125 M glycine for 5 min. Cells were washed twice with ice-cold PBS and lysed with cell lysis buffer (50 mM HEPES (pH7.4), 1 mM EDTA, 85 mM KCl, 10% glycerol, 0.5% NP40, supplemented with protease inhibitor cocktail). Nuclei was collected by centrifugation at 850g for 5 min and suspended in Nuclei lysis buffer (50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 5% glycerol, 1% Triton-X-100, 0.1% SDS, supplemented with protease inhibitor cocktail). The chromatin was sheared by sonication (Branson digital sonicator) to an average size 500 bp and precleared with Protein G PLUS-Agarose (Santa Cruz Biotechnology) or Dynabeads protein G (Life Technologies). The precleared chromatin was incubated with 5 mg of primary antibodies overnight at 4 C, followed by incubation with

Protein G PLUS-Agarose or Dynabeads protein G for 2 h. The immunoprecipitates were washed two times with ChIP wash buffer I (20 mM Tris-HCl (pH 8.0),150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 2 mM EDTA), two times with ChIP wash buffer II (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1% Triton-X-100, 0.1% SDS, 2 mM EDTA), one time with ChIP wash buffer III (20 m Tris-HCl (pH 8.0), 150 mM NaCl, 500 mM LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA) and two times with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). The precipitated chromatin complexes were eluted in elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 30 min with vortex every 5 min. After reverse crosslink at 65 C for 18 h and protein digestion with Proteinase K (Thermo Scientic) at 55 C for 3 h, DNA was isolated using MiniElute PCR purication kit (Quiagen) with 30 ml elution with UltraPure DNAase/RNAase-Free Distilled Water (Life

Technologies). PCR was performed with primers shown in Supplementary Table 2.

Re-ChIP. Re-ChIP assay was performed as described previously34. In brief, crosslinking was performed using 2 mM disuccinimidyl glutarate (Thermo Scientic) for 45 min followed by 1% formaldehyde for 10 min at room temperature. After the rst IP, immune complexes were eluted in Re-ChIP elution buffer (10 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2% SDS, 15 mM DTT) including protease inhibitor cocktail at room temperature for 30 min. The elution was diluted 20 times with ChIP dilution buffer (16.7 mM Tris-HCl (pH 8.0), 167 mM NaCl,1.2 mM EDTA, 1.1% Triton-X) supplemented with protease inhibitor cocktail, 100 mg ml 1 bovine serum albumin , 100 mg ml 1 salmon sperm DNA, followed by second IP. The second IP samples were washed, eluted and puried as described above ChIP procedure. PCR was performed with 1 ml puried DNA by using

PrimeSTAR Max DNA polymerase (Takara) with primers IRAK-M 300 bp

forward and IRAK-M 0 bp reverse shown in Supplementary Table 2.

Statistical analysis. Data are shown as means.d. Statistical analysis was assessed by t-test. Po0.05 was considered statistically signicant.

References

1. Quax, R. A. et al. Glucocorticoid sensitivity in health and disease. Nat. Rev. Endocrinol. 9, 670686 (2013).

2. Clark, A. R. & Belvisi, M. G. Maps and legends: the quest for dissociated ligands of the glucocorticoid receptor. Pharmacol. Ther. 134, 5467 (2012).

3. Beck, I. M. et al. Crosstalk in inammation: the interplay of glucocorticoid receptor-based mechanisms and kinases and phosphatases. Endocr. Rev. 30, 830882 (2009).

4. Luecke, H. F. & Yamamoto, K. R. The glucocorticoid receptor blocks P-TEFb recruitment by NFkappaB to effect promoter-specic transcriptional repression. Genes Dev. 19, 11161127 (2005).

5. Busillo, J. M. & Cidlowski, J. A. The ve Rs of glucocorticoid action during inammation: ready, reinforce, repress, resolve, and restore. Trends Endocrinol. Metab. 24, 109119 (2013).

6. Kadmiel, M. & Cidlowski, J. A. Glucocorticoid receptor signaling in health and disease. Trends Pharmacol. Sci. 34, 518530 (2013).

7. Surjit, M. et al. Widespread negative response elements mediate direct repression by agonist-liganded glucocorticoid receptor. Cell 145, 224241 (2011).

8. Uhlenhaut, N. H. et al. Insights into negative regulation by the glucocorticoid receptor from genome-wide proling of inammatory cistromes. Mol. Cell 49, 158171 (2013).

9. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373384 (2010).

10. Kondo, T., Kawai, T. & Akira, S. Dissecting negative regulation of Toll-like receptor signaling. Trends Immunol. 33, 449458 (2012).

11. von Bernuth, H. et al. Pyogenic bacterial infections in humans with MyD88 deciency. Science 321, 691696 (2008).

12. Adachi, O. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, 143150 (1998).

13. Janssens, S. & Beyaert, R. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol. Cell 11, 293302 (2003).

14. Hassan, F. et al. Involvement of interleukin-1 receptor-associated kinase (IRAK)-M in toll-like receptor (TLR) 7-mediated tolerance in RAW 264.7 macrophage-like cells. Cell. Immunol. 256, 99103 (2009).

15. Deng, J. C. et al. Sepsis-induced suppression of lung innate immunity is mediated by IRAK-M. J. Clin. Invest. 116, 25322542 (2006).

16. Kobayashi, K. et al. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110, 191202 (2002).

17. Berglund, M. et al. IL-1 receptor-associated kinase M downregulates DSS-induced colitis. Inamm. Bowel Dis. 16, 17781786 (2010).

18. Standiford, T. J. et al. TGF-beta-induced IRAK-M expression in tumor-associated macrophages regulates lung tumor growth. Oncogene 30, 24752484 (2011).

19. Sumpter, T. L. et al. DAP12 promotes IRAK-M expression and IL-10 production by liver myeloid dendritic cells and restrains their T cell allostimulatory ability. J. Immunol. 186, 19701980 (2011).

20. van der Windt, G. J. et al. Interleukin 1 receptor-associated kinase m impairs host defense during pneumococcal pneumonia. J. Infect. Dis. 205, 18491857 (2012).

21. Wesche, H. et al. IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family. J. Biol. Chem. 274, 1940319410 (1999).

22. Lagler, H. et al. TREM-1 activation alters the dynamics of pulmonary IRAK-M expression in vivo and improves host defense during pneumococcal pneumonia. J. Immunol. 183, 20272036 (2009).

23. Punturieri, A., Copper, P., Polak, T., Christensen, P. J. & Curtis, J. L. Conserved nontypeable Haemophilus inuenzae-derived TLR2-binding lipopeptides synergize with IFN-beta to increase cytokine production by resident murine and human alveolar macrophages. J. Immunol. 177, 673680 (2006).

24. Wieland, C. W. et al. The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable haemophilus inuenzae from the mouse lung. J. Immunol. 175, 60426049 (2005).

25. Decramer, M., Janssens, W. & Miravitlles, M. Chronic obstructive pulmonary disease. Lancet 379, 13411351 (2012).

26. Sethi, S. & Murphy, T. F. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N. Engl. J. Med. 359, 23552365 (2008).

27. Shuto, T. et al. Activation of NF-kappa B by nontypeable Hemophilus inuenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha /beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells. Proc. Natl Acad. Sci. USA 98, 87748779 (2001).

28. Watanabe, T., Jono, H., Han, J., Lim, D. J. & Li, J. D. Synergistic activation of NF-kappaB by nontypeable Haemophilus inuenzae and tumor necrosis factor alpha. Proc. Natl Acad. Sci. USA 101, 35633568 (2004).

29. Jono, H. et al. NF-kappaB is essential for induction of CYLD, the negative regulator of NF-kappaB: evidence for a novel inducible autoregulatory feedback pathway. J. Biol. Chem. 279, 3617136174 (2004).

30. Dhawan, L., Liu, B., Blaxall, B. C. & Taubman, M. B. A novel role for the glucocorticoid receptor in the regulation of monocyte chemoattractant protein-1 mRNA stability. J. Biol. Chem. 282, 1014610152 (2007).

31. Gille, J., Reisinger, K., Westphal-Varghese, B. & Kaufmann, R. Decreased mRNA stability as a mechanism of glucocorticoid-mediated inhibition of vascular endothelial growth factor gene expression by cultured keratinocytes.J. Invest. Dermatol. 117, 15811587 (2001).32. Robertson, S. et al. Abrogation of glucocorticoid receptor dimerization correlates with dissociated glucocorticoid behavior of compound a. J. Biol. Chem. 285, 80618075 (2010).

33. Baldwin, Jr. A. S. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649683 (1996).

34. Truax, A. D. & Greer, S. F. ChIP and Re-ChIP assays: investigating interactions between regulatory proteins, histone modications, and the DNA sequences to which they bind. Methods Mol. Biol. 809, 175188 (2012).

35. Suzuki, N. et al. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 416, 750756 (2002).

36. Picard, C. et al. Pyogenic bacterial infections in humans with IRAK-4 deciency. Science 299, 20762079 (2003).

NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7062

37. Scheinman, R. I., Gualberto, A., Jewell, C. M., Cidlowski, J. A. & Baldwin, Jr. A.S. Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol. Cell. Biol. 15, 943953 (1995).38. Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A. & Karin, M. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 270, 286290 (1995).

39. Kassel, O. et al. Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J. 20, 71087116 (2001).

40. Oda, K. & Kitano, H. A comprehensive map of the toll-like receptor signaling network. Mol. Syst. Biol. 2, 0015 (2006).

41. Li, F., Thiele, I., Jamshidi, N. & Palsson, B. O. Identication of potential pathway mediation targets in Toll-like receptor signaling. PLoS Comput. Biol. 5, e1000292 (2009).

42. Nissen, R. M. & Yamamoto, K. R. The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 14, 23142329 (2000).

43. Rao, N. A. et al. Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes. Genome Res. 21, 14041416 (2011).

44. Su, J., Xie, Q., Wilson, I. & Li, L. Differential regulation and role of interleukin-1 receptor associated kinase-M in innate immunity signaling. Cell. Signal. 19, 15961601 (2007).

45. Cabanski, M. et al. Genome-wide transcriptional proling of mononuclear phagocytes recruited to mouse lungs in response to alveolar challenge with the TLR2 agonist Pam3CSK4. Am. J. Physiol. Lung Cell. Mol. Physiol. 297, L608L618 (2009).

46. Nakayama, K. et al. Involvement of IRAK-M in peptidoglycan-induced tolerance in macrophages. J. Biol. Chem. 279, 66296634 (2004).

47. Zacharioudaki, V. et al. Adiponectin promotes endotoxin tolerance in macrophages by inducing IRAK-M expression. J. Immunol. 182, 64446451 (2009).

48. Li, H. et al. IL-1 receptor-associated kinase M is a central regulator of osteoclast differentiation and activation. J. Exp. Med. 201, 11691177 (2005).

49. Balaci, L. et al. IRAK-M is involved in the pathogenesis of early-onset persistent asthma. Am. J. Hum. Genet. 80, 11031114 (2007).

50. Seki, M. et al. Critical role of IL-1 receptor-associated kinase-M in regulating chemokine-dependent deleterious inammation in murine inuenza pneumonia. J. Immunol. 184, 14101418 (2010).

51. van t Veer, C. et al. Induction of IRAK-M is associated with lipopolysaccharide tolerance in a human endotoxemia model. J. Immunol. 179, 71107120 (2007).

52. Medzhitov, R., Schneider, D. S. & Soares, M. P. Disease tolerance as a defense strategy. Science 335, 936941 (2012).

53. Tournoy, K. G., Kips, J. C., Schou, C. & Pauwels, R. A. Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness. Clin. Exp. Allergy 30, 7985 (2000).

54. Kwak, Y. G. et al. Involvement of PTEN in airway hyperresponsiveness and inammation in bronchial asthma. J. Clin. Invest. 111, 10831092 (2003).55. Matute-Bello, G. et al. An ofcial American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am. J. Respir. Cell Mol. Biol. 44, 725738 (2011).

56. Hu, H. L., Zhang, Q., Kong, B. & Shi, X. Expression of pancreatic regenerating gene in lung and intestinal tissue correlates with the severity of disease in rats with acute necrotizing pancreatitis. Mol. Med. Rep. 7, 503508 (2013).

57. Erwin, A. L. & Smith, A. L. Nontypeable Haemophilus inuenzae: understanding virulence and commensal behavior. Trends Microbiol. 15, 355362 (2007).

58. Yoshida, H., Jono, H., Kai, H. & Li, J. D. The tumor suppressor cylindromatosis (CYLD) acts as a negative regulator for toll-like receptor 2 signaling via negative cross-talk with TRAF6 AND TRAF7. J. Biol. Chem. 280, 4111141121 (2005).

59. Lim, J. H. et al. CYLD negatively regulates transforming growth factor-beta-signalling via deubiquitinating Akt. Nat. Commun. 3, 771 (2012).

60. Komatsu, K. et al. Inhibition of PDE4B suppresses inammation by increasing expression of the deubiquitinase CYLD. Nat. Commun. 4, 1684 (2013).

61. Mercurio, F. et al. IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation. Science 278, 860866 (1997).

62. Ishinaga, H. et al. TGF-beta induces p65 acetylation to enhance bacteria-induced NF-kappaB activation. EMBO J. 26, 11501162 (2007).

63. Johannessen, C. M. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968972 (2010).

64. Wang, J. C. et al. Chromatin immunoprecipitation (ChIP) scanning identies primary glucocorticoid receptor target genes. Proc. Natl Acad. Sci. USA 101, 1560315608 (2004).

Acknowledgements

This work was supported by grants from the National Institutes of Health DC005843,

DC004562 and GM107529 to J.D.L. J.D.L. is a Georgia Research Alliance Eminent

Scholar in Inammation and Immunity. R.A.F. is a Howard Hughes Medical Institute

(HHMI) investigator.

Author contributions

M.M., J.-Y.L., S.S.-M., H.X. and J.-D.L. designed the experiments and analysed the data.

M.M., J.-Y.L. and S.S.-M. performed the experiments. H.K. contributed to data analysis

and discussion. R.A.F. and K.S.K. contributed materials and also contributed to

discussion. M.M., W.Y.W. and J.-D.L. wrote the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/

http://www.nature.com/naturecommunications

Web End =naturecommunications

Competing nancial interests: The authors declare no competing nancial interests.

Reprints and permission information is available online at http://npg.nature.com/reprintsand

Web End =http://npg.nature.com/

http://npg.nature.com/reprintsand

Web End =reprintsand permissions/

How to cite this article: Miyata, M. et al. Glucocorticoids suppress inammation

via the upregulation of negative regulator IRAK-M. Nat. Commun. 6:6062

doi: 10.1038/ncomms7062 (2015).

This work is licensed under a Creative Commons Attribution 4.0

International License. The images or other third party material in this

article are included in the articles Creative Commons license, unless indicated otherwise

in the credit line; if the material is not included under the Creative Commons license,

users will need to obtain permission from the license holder to reproduce the material.

To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/

12 NATURE COMMUNICATIONS | 6:6062 | DOI: 10.1038/ncomms7062 | http://www.nature.com/naturecommunications

Word count: 10129

Show less

Abstract

Translate

Glucocorticoids are among the most commonly used anti-inflammatory agents. Despite the enormous efforts in elucidating the glucocorticoid-mediated anti-inflammatory actions, how glucocorticoids tightly control overactive inflammatory response is not fully understood. Here we show that glucocorticoids suppress bacteria-induced inflammation by enhancing IRAK-M, a central negative regulator of Toll-like receptor signalling. The ability of glucocorticoids to suppress pulmonary inflammation induced by non-typeable Haemophilus influenzae is significantly attenuated in IRAK-M-deficient mice. Glucocorticoids improve the survival rate after a lethal non-typeable Haemophilus influenzae infection in wild-type mice, but not in IRAK-M-deficient mice. Moreover, we show that glucocorticoids and non-typeable Haemophilus influenzae synergistically upregulate IRAK-M expression via mutually and synergistically enhancing p65 and glucocorticoid receptor binding to the IRAK-M promoter. Together, our studies unveil a mechanism by which glucocorticoids tightly control the inflammatory response and host defense via the induction of IRAK-M and may lead to further development of anti-inflammatory therapeutic strategies.

Details

Title

Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

Author

Miyata, Masanori; Lee, Ji-yun; Susuki-miyata, Seiko; Wang, Wenzhuo Y; Xu, Haidong; Kai, Hirofumi; Kobayashi, Koichi S; Flavell, Richard A; Li, Jian-dong

Pages

6062

Publication year

2015

Publication date

Jan 2015

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms7062

ProQuest document ID

1645224737

Glucocorticoids suppress inflammation via the upregulation of negative regulator IRAK-M

Jump to:

Full text

Abstract

Details

Suggested sources