IAI Accepts, published online ahead of print on 22 September 2014 Infect. Immun. doi:10.1128/IAI.02546-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

TLR triggered ATP release enhances antibacterial immunity

1

TLR triggered calcium mobilization protects mice against bacterial infection through

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extracellular ATP release

3

Hua Ren, Yunfei Teng, Binghe Tan, Xiaoyu Zhang, Wei Jiang, Mingyao Liu,

4

Wenzheng Jiang, Bing Du*, Min Qian*

5

Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical

6

Sciences and School of Life Sciences, East China Normal University,

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Shanghai, China

8 9

Running Title: TLR triggered ATP enhances antibacterial immunity

10 11

* To whom correspondence should be addressed

12 13

Corresponding author: Min Qian, Institute of Biomedical Sciences and

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School of Life Sciences, East China Normal University, 500 Dongchuan Road,

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Shanghai 200241, China; Tel: +86-21-54341039; Fax: +86-21-54344922;

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E-mail: [email protected]

17

Bing Du, Institute of Biomedical Sciences and School of Life Sciences, East

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China Normal University, 500 Dongchuan Road, Shanghai 200241, China; Tel:

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+86-21-24206964; Fax: +86-21-54344922; E-mail: [email protected]

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1

TLR triggered ATP release enhances antibacterial immunity

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Abstract

22

Extracellular ATP (eATP) released as a “danger signal” by injured or stressed

23

cells plays an important role in the regulation of immune responses. But the

24

relationship between ATP release and innate immune responses is still

25

uncertain. Here, we demonstrated that ATP was released through

26

TLR-associated signaling in both E. coli infected mice and LPS or Pam3CSK4

27

treated macrophages. This ATP release could be completely blocked only by

28

N-ethylmaleimide (NEM) but not by carbenoxolone (CBX), flufenamic acid

29

(FFA) or Probenecid, suggesting the key role of exocytosis in this process.

30

Furthermore, LPS-induced ATP release could also be dramatically reduced

31

through

32

thapsigargin (TG). In addition, the secretion of IL-1β and CCL-2 was enhanced

33

significantly by ATP in a time and dose dependent manner. Meanwhile,

34

macrophage mediated phagocytosis of bacteria was also promoted

35

significantly by ATP stimulation. Furthermore, extracellular ATP reduces the

36

invading bacteria and protects mice from peritonitis through activating

37

purinergic receptors. Mechanistically, phosphorylation of AKT and ERK was

38

overtly increased by ATP in anti-bacterial immune responses. Accordingly, if

39

we blocked the P2X and P2Y associated signaling pathway by Suramin and

40

pyridoxalphosphate-6-azophenyl-2',4'-disulfonic

41

(PPADS), the ATP enhanced-immune response was restrained significantly.

42

Taken together, our study reveals an internal relationship between danger

suppressing

calcium

mobilization

by

U73122,

acid

caffeine

tetrasodium

and

salt

2

TLR triggered ATP release enhances antibacterial immunity

43

signal and TLR signaling in innate immune responses which suggests a

44

potential therapeutic significance of calcium mobilization mediated ATP

45

release in infectious diseases.

46

Key words: ATP, TLR, exocytosis, calcium mobilization, danger signal

47

3

TLR triggered ATP release enhances antibacterial immunity

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Introduction

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The concept of purinergic transmission via ATP was first proposed by Burnstock in

50

1972 (1). Actually, extracellular nucleotides arising from non-neuronal sources are

51

also involved in many important physiological and pathological processes such as cell

52

injury, hypoxia, inflammation and even tumorigenesis (2, 3). Under normal conditions,

53

cytosolic concentrations of ATP range from 3 to 10 mM, whereas eATP varies widely,

54

from pmol to μmol, in different cells and tissues (4). When cells were stressed,

55

cytosolic ATP was released from damaged cells and alarmed immune cells through

56

purinergic receptors in an autocrine or paracrine manner (5). Thus, the nucleotides

57

released from damaged or infected cells can also be considered a “Danger signal” to

58

alert the immune system through activating purinergic receptors.

59

Generally, injured or infected cells can release ATP through the cytomembrane in

60

different ways. Early studies indicated ATP-binding cassette (ABC) transporters such

61

as cystic fibrosis transmembrane regulator (CFTR) regulated the release of ATP

62

through ion channels (6). Recent studies have suggested gap junction, membrane

63

channels, hemichannels such as connexin and pannexin hemichannels (7, 8),

64

maxi-anion channels (9, 10), volume-regulated anion channels (11), and P2 receptors

65

are all involved in ATP release (12). In addition to release through channels,

66

non-excitatory cells can release ATP by exocytotic mechanisms in response to

67

biochemical and mechanical stimuli, such as neurons upon depolarization (13-15).

68

The mechanisms of ATP release are well characterized in vesicular cells and

69

excitatory/secretory tissues; however, the mechanisms of ATP release from immune 4

TLR triggered ATP release enhances antibacterial immunity

70

cells such as macrophages have not been well characterized. Actually, a recent paper

71

showed that ATP could be released from infected immune cells and immune responses

72

upregulated through activating NLRP3 inflammasomes, but the correlation between

73

ATP release and TLR signaling is still indistinct (16). Therefore, the exploration of the

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internal relationship between ATP release and TLR-mediated innate immune

75

responses against bacterial infection is justified and worthwhile.

76

As the first defense against invading pathogens, the innate immune system can

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recognize pathogen-associated molecular patterns (PAMPs) by germline-encoded

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pattern-recognition receptors (PRRs) in a non-specific but instantaneous manner (17).

79

Among them, endocytic pattern-recognition receptors mainly promote engulfment and

80

destruction of microorganisms by phagocytes, whereas signaling pattern-recognition

81

receptors such as Toll-like receptors (TLR), NOD-like receptors (NLR) and

82

RIG-I-like receptors (RLR) trigger the synthesis and secretion of immune regulators

83

or molecules such as cytokines and chemokines that are crucial in regulating innate

84

and adaptive immunity (18-20). The activation of TLRs by given agonists induces the

85

secretion of proinflammatory cytokines, chemokines, and type I interferon, such as

86

TNF-α, IL-6, MCP-1 and IFN-β, to fight against invading pathogens and recruit

87

peripheral lymphocytes to the site of infection (21). In the past decades, most studies

88

have focused on the secretion of cytokines, chemokines and interferon in

89

TLR-mediated immune responses. Actually, extracellular nucleotides secreted by

90

injured or infected cells also play important roles in the innate immune system.

91

Understanding the cross-talk between TLR and purinergic signaling represents a 5

TLR triggered ATP release enhances antibacterial immunity

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novel approach to better understand immune regulation.

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In this study, we demonstrated that ATP was released from immune cells during

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bacterial infection mainly through exocytosis by TLR-dependent intracellular calcium

95

mobilization. Then, eATP facilitated macrophage mediated phagocytosis and cytokine

96

production to clear invading bacteria through activating P2 receptors. Furthermore,

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we also provide evidence that ATP has a non-redundant role in promoting host

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survival from E. coli induced peritonitis. TLR signaling not only can be involved in

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inflammation responses through the typical MAPK pathway but can also regulate

100

innate immune responses via increasing extracellular “Danger signals” such as eATP.

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Taken together, our study has shown a novel role of TLR-triggered calcium

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mobilization in the upregulation of innate immune responses against bacterial

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infection through extracellular ATP release.

104 105

Materials and Methods

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Animals

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For peritoneal macrophage (PM) and bone marrow macrophage (BMM) isolation, as

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well as the peritonitis mouse model, female 6–8 week old C57BL/6 mice were

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purchased from the Shanghai Laboratory Animal Company (Shanghai, China). All

110

mice used in these experiments were housed under pathogen-free conditions and were

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maintained in accordance with institutional guidelines. All experimental protocols

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were approved by the Animal Investigation Committee of East China Normal

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University. 6

TLR triggered ATP release enhances antibacterial immunity

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Cell culture

115

BMMs and PMs were obtained as previously described (22). The mouse macrophage

116

cell line RAW 264.7 was obtained from the American Type Culture Collection

117

(Rockville, MD). BMMs, PMs and RAW 264.7 were maintained in DMEM

118

containing 10% fetal bovine serum, 100 units/ml penicillin and 100 mg/ml

119

streptomycin, 5% CO2, 95% humidity until drugs were added at the times indicated.

120

ATP assay

121

RAW 264.7 cells (5x104 cells/well) were loaded in 24-well plates (Corning Costar,

122

Corning, NY) with DMEM containing 10% fetal bovine serum for 24 h, then starved

123

in serum free DMEM for 12 h before ATP assay. BMMs (1 × 105 cells/well) and PMs

124

(1× 105 cells/well) were cultured in FBS-free DMEM for 12 h. Then, RAW 264.7 cells,

125

BMMs and PMs were maintained in 500 μl fresh serum free DMEM. LPS from

126

Escherichia coli 055:B5 and Pam3CSK4 (InvivoGen, San Diego, CA) and other

127

inhibitors were added at the times indicated. At specific time points, supernatants

128

were collected and used to quantify extracellular ATP using the ATP Bioluminescent

129

Assay Kit (Sigma-Aldrich, St. Louis, MO) as recommended by the manufacturer. ATP

130

levels were calculated based on an ATP standard curve. For ATP detection in vivo,

131

bacterial strain Escherichia coli 0111:B4 was injected into the mouse abdominal

132

cavity for different time periods. One milliliter of sterile PBS was used to wash out

133

the peritoneal fluid from mice sacrificed by cervical dislocation at different times.

134

Peritoneal fluid ATP was assayed as described above. All samples were set up in

135

triplicate and all experiments were repeated at least three times. 7

TLR triggered ATP release enhances antibacterial immunity

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Measurement of Ca2+ mobilization

137

RAW 264.7 cells were grown on 20-mm-diameter glass bottom dishes (NEST

138

Biotechnology, Jiangsu, China) in complete DMEM containing 10% FBS, 100

139

units/ml penicillin and 100 mg/ml streptomycin. Before Ca2+ imaging, the medium

140

was replaced with HBSS buffer (145 mM NaCl, 20 mM HEPES, 2.5 mM KCl, 1mM

141

MgCL2.6H2O, 1.8 mM CaCl2, 10 mM glucose, and 0.01% BSA, pH 7.4) and loaded

142

with Furo-2/ AM (2 μM) (Beyotime, Jiangsu, China) for 30 minutes at room

143

temperature in the dark. After dye loading, the cells were washed with HBSS buffer

144

three times and then treated with the appropriate inhibitor. Fura-2 fluorescence was

145

imaged at 340 and 380 nm excitation to detect intracellular free calcium (Olympus

146

IX71 and LAMBDA DG-4, Novato, CA, USA) and recorded by InVivo software and

147

then analyzed by Image-Pro Analyzer 6.2 (Media Cybernetics, Bethesda, MD, USA).

148

RNA isolation and RT-PCR

149

BMMs, PMs and RAW 264.7 cells were stimulated with different concentrations of

150

ATP (Sigma-Aldrich, St. Louis, MO) or LPS for 1 h and total RNA was isolated by

151

applying TRIzol reagent (Invitrogen) according to the protocol of the manufacturer.

152

cDNA was synthesized with 100 ng RNA using a reverse transcription kit (Prime

153

Script 1st Strand cDNA Synthesis kit (TAKARA, Dalian, China) according to the

154

manufacturer’s instructions. One microliter of template from 10-fold diluted cDNA

155

was subjected to quantification of cytokine expression using iQ SYBR Green

156

Supermix (Bio-Rad, Hercules, CA), and the data were analyzed by the ECO™ Real

157

Time PCR System (Illumina,San Diego, CA). The sequence-specific primers are 8

TLR triggered ATP release enhances antibacterial immunity

158

shown in table I/II.

159

ELISA assay

160

To assay secreted CCL-2 and IL-1β, RAW 264.7 cells or PMs were seeded in a 96

161

well plate (Corning Costar, Corning, NY) at 5 × 104/well and cultured in DMEM

162

containing 10% FBS overnight. Then, the cells were pretreated with drugs as

163

indicated. After stimulation with ATP for an additional 6 h using PBS as a negative

164

control, the supernatants were centrifuged for 5 min. Secretion of IL-1β and CCL-2

165

was measured by Mouse OptEIA ELISA kits (BD Biosciences, San Diego, CA).

166

Phagocytosis

167

For fluorescence labeled bacteria, E. coli BL21 with plasmid pET24a-GFP were

168

grown in Luria-Bertani broth (Difco) at 37 ℃ to OD450 at 0.6 and induced for GFP

169

expression by IPTG at 16 ℃ overnight. Bacteria were collected and washed by

170

sterile PBS for bacteria number determination. RAW 264.7 cells were pretreated with

171

or without purinergic receptor inhibitors, and then stimulated with ATP for 30 minutes.

172

Subsequently, E. coli-GFP and RAW 264.7 cells were cultured together at a ratio of

173

five colony forming units (CFU) of E. coli per cell at 37 ℃. After 30 minutes,

174

external bacteria were removed by washing three times with PBS and the cells were

175

incubated for one minute in 0.4 % trypan blue to quench extracellular GFP. After a

176

final wash in PBS, the cells were collected and resuspended in PBS to determine the

177

efficiency of phagocytosis on a FACScan flow cytometer (BD Biosciences, San Diego,

178

CA).

179

Transwell migration assay 9

TLR triggered ATP release enhances antibacterial immunity

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After starving in serum free DMEM overnight, RAW 264.7 cells (1 × 105) suspended

181

in serum free DMEM were added into the upper well of a 24 well transwell insert

182

with 8.0 μm polycarbonate membrane (Corning, Glendale, Ariz) and the bottom well

183

was supplemented with DMEM containing 10% FBS with ATP or ATPγS. After 8 h of

184

migration, the insert membrane was scraped gently with a cotton swab to remove

185

residual cells, then migrated cells in the lower membrane were fixed with 4%

186

paraformaldehyde. Finally, Giemsa staining was conducted to count the cells from

187

five randomly fields at x200 magnification using an optical microscope.

188

Western blotting

189

BMMs were seeded in 6-well plates (Corning Costar, Corning, NY) and stimulated

190

with ATP at the times indicated. Samples were separated by 12% SDS-PAGE and

191

transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA).

192

After incubation with phosphor-ERK1/2, ERK1/2, phosphor-AKT, AKT, β-actin,

193

phosphor-p38, p38 and IKB-α antibody (Cell Signaling Technology, Danvers, MA),

194

the PVDF membranes were incubated with appropriate HRP-conjugated secondary

195

antibodies (Sigma-Aldrich, St. Louis, MO). Finally, the ECL (Rockford, IL) method

196

was applied to detect those proteins.

197

Peritonitis mouse model

198

Six-to-eight week old C57BL/6 female mice were chosen to induce peritonitis to

199

mimic bacterial infection. Peritoneal bacteria and survival curves were obtained to

200

reflect the protective effect of ATP. To count peritoneal fluid E.coli, mice were divided

201

randomly into seven groups and pretreated with an intraperitoneal injection of PBS, 10

TLR triggered ATP release enhances antibacterial immunity

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ATP (50 mg/kg), apyrase (100 U/kg; Sigma-Aldrich, St. Louis, MO), Pyridoxal

203

phosphate-6-azo (benzene-2,4-disulfonic acid), tetrasodium salt hydrate (PPADS, 6

204

mg/kg; Sigma-Aldrich, St. Louis, MO), Suramin hexasodium salt (Suramin, 75 mg/kg;

205

Sigma-Aldrich, St. Louis, MO), N-ethylmaleimide (NEM,15 mg/kg; Sigma-Aldrich,

206

St. Louis, MO), or U73122 (120 ng/kg; Sigma-Aldrich, St. Louis, MO). Twelve hours

207

later, each mouse was challenged with E. coli 0111:B4 through intraperitoneal

208

injection. After 12 hours, E.coli were lavaged with 2 ml PBS from each mouse’s

209

abdominal cavity, then diluted 10-fold in PBS, and 20 μl of the bacterial suspension

210

was cultured in LB solid medium for 12 h. Single CFU were counted to determine

211

peritoneal fluid E.coli. Another seven groups were divided and treated as described

212

above, but instead of counting peritoneal fluid E.coli, the mice were checked every

213

two hours to obtain a survival curve.

214

Statistical analysis

215

Data are presented as means ± SEMs (n= 3-6). Statistical significance was evaluated

216

with the Student’s t-test or one-way ANOVA followed by Dunnertt’s multiple

217

comparison. P values ൏ 0.05 and ൏ 0.01 were considered significant and very

218

significant, respectively. For survival curve analysis, the log-rank test was performed

219

and a p value ൏0.05 was considered significant.

220 221

Results

222

ATP is released from macrophages during bacterial infection. To elucidate the role

223

of eATP in anti-bacterial immune responses, we measured the concentration of eATP 11

TLR triggered ATP release enhances antibacterial immunity

224

using an ATP bioluminescent assay kit in both E. coli infected mice and LPS or

225

Pam3CSK4 treated macrophages in a time-dependent manner. As shown in Fig.1 A,

226

eATP in the peritoneal cavity of mice challenged with E. coli 0111:B4 was

227

dramatically increased in 30 minutes. Then the eATP returned to a relatively low level

228

as time passed in the following 6 h. Furthermore, a similar ATP release was also

229

observed in LPS treated BMMs, PMs and RAW 264.7 cells (Fig. 1B). In order to

230

further explore the role of TLR signaling in bacteria-induced ATP release, we

231

challenged RAW 264.7 cells with LPS and Pam3CSK4, which are specific agonists of

232

TLR4 and TLR2, respectively. Interestingly, ATP release was increased both by LPS

233

and Pam3CSK4 in RAW 264.7 cells, which implied an internal relation between TLR

234

signaling and ATP secretion (Fig. 1C). In order to further explore the potential role of

235

purinergic signaling in LPS-mediated immune responses, we treated the RAW 264.7

236

cells with both ATP and LPS to detect the expression levels of ATP associated

237

receptors. As shown in Fig. 1D, the expression levels of P2X1, P2X4, P2X7 and P2Y2

238

were all overtly increased by LPS. Conversely, the expression of P2X2, P2X3, P2X5,

239

P2X6, P2Y1 and P2Y4 was decreased in each case by LPS, while the expression of

240

these receptors was little influenced by ATP.

241

TLR triggers ATP release through calcium dependent exocytosis. Generally, ATP

242

may

243

volume-regulated anion channels when cells are stressed. So we treated the LPS

244

challenged cells with different inhibitors such as NEM (a specific inhibitor of the

245

soluble

be

released

N-ethyl

through

membrane

maleimide-sensitive

channels,

factor

maxi-anion

attachment

channels

protein

and

receptor), 12

TLR triggered ATP release enhances antibacterial immunity

246

carbenoxolone

(CBX,

a

non-specific

pannexin

channel

inhibitor),

247

18-alpha-glycyrrhetinic acid (18AGA), flufenamic acid (a gap junction inhibitor) and

248

probenecid (a selective pannexin-1 channel inhibitor). As shown in Fig.2 A,

249

LPS-induced ATP release was significantly inhibited by NEM but not by the other

250

inhibitors, suggesting a key role of exocytosis in LPS-mediated ATP release. It is well

251

known that ER stress induced calcium release potentiated ER to Golgi apparatus

252

exocytosis that facilitated ATP release from vesicular transport. However, brefeldin A

253

(BFA) and chloroquine (CQ) did not affect ATP release during bacterial infection (Fig.

254

2B). Meanwhile, we blocked LPS-activated signaling with U73122 (phospholipase c,

255

PLC inhibitor), caffeine and thapsigargin (TG) (both were used to drain intracellular

256

Ca2+ from the ER) to explore the mechanism of LPS-associated ATP release. Similar

257

to the above data, ATP release was activated by LPS, and this activation could be

258

dramatically inhibited by U73122, caffeine and TG. These data suggested that

259

LPS-induced ATP release is strongly associated with calcium mobilization. So we

260

further appraised the effects of U73122, caffeine and TG on LPS-induced calcium

261

mobilization (Fig. 2C). Calcium mobilization was increased in LPS-treated cells and

262

could be blocked almost entirely blocked by these inhibitors. ATP release through

263

exocytosis is dependent on intracellular Ca2+ mobilization, but extracellular Ca2+

264

effluence is not necessary as application of CaCl2 did not affect ATP release (Fig. 2B).

265

These results implied that LPS induced ATP release mainly through PLC-dependent

266

calcium mobilization and calcium mediated exocytosis.

267

ATP enhances host defense against invading bacteria in a peritonitis mouse 13

TLR triggered ATP release enhances antibacterial immunity

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model. To validate the role of eATP in promoting host defense against microbes, we

269

set up a mouse peritonitis model by intraperitoneal injection of E. coli 0111:B4 to

270

monitor the clearance of bacteria and mouse survival. As shown in Fig. 3A, the total

271

quantity of bacteria in the peritoneal cavity was reduced in ATP injected mice. In

272

contrast, if we treated the mice with apyrase to degrade endogenous ATP, the invading

273

bacteria were little changed. After using U73122 and NEM to inhibit ATP release, the

274

total quantity of bacteria increased significantly compared to PBS-treated mice.

275

Accordingly, the survival of ATP treated mice was also significantly extended when

276

compared with control, apyrase, NEM and U73122 treated groups (Fig. 3B),

277

suggesting the key role of eATP in the clearance of invading bacteria.

278

ATP increases IL-1β and CCL-2 production through P2 receptors. The activation

279

of P2 receptors by ATP is the main step in its regulation of inflammation and immune

280

responses. Thus, we checked the expression of P2 receptors in murine macrophages

281

by real time PCR (Fig. 4A). Almost all ATP-associated P2 receptors were expressed in

282

PMs and RAW 264.7 cells, but their abundance varied widely. Among them, P2X7

283

and P2Y2 were the most abundant receptors. As shown in Fig. 4B, IL-1β and CCL-2

284

mRNAs were both increased dramatically by ATP in a time dependent manner but not

285

those for other inflammatory cytokines such as TNF-α, IL-6, iNOS, COX-2 and

286

IFN-β. The expression and secretion of IL-1β and CCL-2 could also be activated by

287

ATP in a dose-dependent manner that could be eliminated by apyrase through

288

degradation of ATP in BMMs and PMs (Fig. 4C~F). To further explore the

289

mechanism of ATP-facilitated IL-1β and CCL-2 production, we also treated the cells 14

TLR triggered ATP release enhances antibacterial immunity

290

with the P2 receptor inhibitors PPADS and Suramin. As shown in Fig. 4G&H, the

291

ATP-mediated increases in IL-1β and CCL-2 production were both blocked by

292

Suramin and PPADS, suggesting the crucial role of P2 receptors in ATP-regulated

293

immune responses.

294

Macrophage-mediated phagocytosis is promoted significantly by ATP. As the key

295

immune cell in the innate immune system, macrophages can engulf and digest

296

pathogens and participate in antigen presentation. So, we evaluated macrophage

297

mediated pathogen phagocytosis and chemotaxis in ATP treated cells. As shown in Fig.

298

5A&B, the phagocytosis of GFP-labeled bacteria by RAW 264.7 cells was increased

299

by pretreating with ATP (100 μM), and the increased phagocytosis could be reduced

300

by apyrase, PPADS and Suramin. Although the secretion of CCL-2 was promoted by

301

ATP, macrophage mediated chemotaxis was little influenced by ATP (data not shown).

302

Taken together, our current data further confirm the positive regulation by ATP of

303

macrophage-mediated phagocytosis.

304

ATP activates the AKT/ERK associated signaling pathway. Transcriptional

305

induction of cytokine or chemokine production requires coordinated and cooperative

306

activation of different transcription factors such as NF-κB, AKT, ERK and p38. Thus,

307

we performed western blotting to determine the signaling pathway influenced in

308

ATP-treated BMMs. As shown in Fig. 6A, ATP stimulation resulted in a time

309

dependent increase in phosphorylation of ERK and AKT, whereas IκBα and p38 were

310

little affected. So, this data implied that ATP enhanced innate immune responses

311

mainly through the AKT/ERK associated signaling pathway. Furthermore, we also 15

TLR triggered ATP release enhances antibacterial immunity

312

treated some cells with the ERK inhibitor U0126, the P2 inhibitor PPADS and

313

Suramin to see the influence of P2 receptors on ATP-induced ERK activation.

314

Consistent with the above data, the phosphorylation of ERK was greatly enhanced by

315

ATP, and this activation could be partially rescued by U0126, PPADS and Suramin.

316

ATP enhances the host defense against bacterial infection through P2 receptors.

317

To investigate the function of P2 receptors in the regulation of innate immunity, we

318

set up an acute peritonitis mouse model and survival was monitored every 2 hours

319

over the next 48 h. As shown in Fig. 6B, the residual bacteria in the abdominal cavity

320

were significantly reduced by pretreating the mice with ATP compared with the

321

control and degraded ATP. Furthermore, the numbers of residual bacteria could even

322

be doubled by treating with Suramin and PPADS when compared with the control

323

group, suggesting the important role of purinergic signaling in clearing invading

324

pathogens. Consequently, the survival of infected mice was also increased

325

dramatically by ATP injection, and ATP-mediated protection could also be eliminated

326

by the P2 receptor inhibitors Suramin and PPADS (Fig. 6C). Thus, our results

327

suggested a critical role for ATP and P2 receptors in regulation of the immune

328

response and pathogen clearance in bacteria-mediated peritonitis.

329 330 331

Discussion

332

As a key “Danger signal”, ATP has been investigated for a long time in terms of the

333

regulation of immune responses. But most studies in the past few decades only 16

TLR triggered ATP release enhances antibacterial immunity

334

focused on the function of ATP and it receptors. Recently, the physiological effect of

335

extracellular ATP on NLRP3 inflammasome activation was investigated in the context

336

of bacterial infection (16). Why and how ATP is released during bacteria infection is

337

still unknown. In this study, we demonstrated that ATP could be released from

338

infected or stressed cells via a calcium dependent manner which was induced by TLR

339

signaling. Then the released ATP could upregulate innate immune responses in

340

macrophages and enhance the host defense against bacterial infection through

341

activating P2X and P2Y receptors. Therefore, we can hypothesize that

342

bacterial-induced TLR signaling not only activates classic immune responses, but also

343

induces ATP release through calcium mobilization, which could also facilitate the

344

immune cells in an autocrine and/or paracrine manner. Actually, the release of ATP

345

seems more important to the immune system because the activation of TLRs can only

346

transduce intracellular signaling in the immune cells, but ATP can easily deliver the

347

alarm signal to neighboring cells in a very short time in a manner which is even

348

quicker and more extensive than cytokine production. So the release of ATP can also

349

be regarded as a kind of feedback regulatory loop that could be important in

350

protecting the host from bacterial infection (Fig. 6D). Our data broaden the

351

understanding of eATP as a danger signal in host defense against bacterial infection,

352

which shows great potential of purinergic signaling in anti-bacterial drug discovery.

353

Innate

354

pathogen-associated molecular patterns (PAMPs) by TLRs and are involved in

355

autoimmune, chronic inflammatory and infectious diseases. Interestingly, extracellular

and

adaptive

immunity

are

activated

through

recognition

of

17

TLR triggered ATP release enhances antibacterial immunity

356

signaling molecules can also bind to specific cell membrane receptors in order to

357

exhibit a unique and important role in cellular communication and signal transduction.

358

In fact, ATP is found not only inside almost every living cell, where it is

359

predominantly known for its central role in cellular energy metabolism, but is also

360

widely distributed outside the cell, where it appears to influence a series of biological

361

processes such as the generation of chemotactic signals and activation of immune

362

cells in response to invading pathogens (23). Thus the continued study of the

363

cross-talk between TLRs and purinergic signaling will be important. As shown in Fig.

364

1C, both LPS and Pam3CSK4 could induce the release of ATP, obviously suggesting

365

an inherent relationship between TLRs and ATP mediated signaling. Interestingly, the

366

release of ATP is not a steady process during bacterial infection. When the bacteria

367

invaded, eATP could reach its highest level in only 30 minutes, and then the eATP

368

was degraded to a relatively low level. This phenomenon implies that the release of

369

ATP must be regulated by a transient mechanism that is similar to the activation of

370

TLR signaling.

371

Generally, ATP and other nucleotides are released mainly through three potential

372

mechanisms: exocytosis, blebbing or passage via a plasma membrane channel (24).

373

So in this case, ATP release may occur through exocytosis during bacterial infection

374

because channel activation requires a relatively long time (25, 26). Consistent with

375

our hypothesis, LPS-induced ATP release was dramatically reduced by NEM but not

376

by inhibitors to plasma membrane channels (CBX, FFA and Probenecid), as shown in

377

Fig. 2A. Although the recognition to the PAMPs is specific to each TLR, individual 18

TLR triggered ATP release enhances antibacterial immunity

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TLRs mainly activate similar transcription factors such as NF-κB, activating protein-1

379

and interferon regulatory factors, driving specific immune responses (27). Meanwhile,

380

LPS can also evoke transient Ca2+ in monocyte/macrophages and astrocytes (28, 29).

381

LPS induces a Ca2+ increase through release from intracellular Ca2+ stores instead of

382

an extracellular Ca2+ efflux (30). It has been reported that intracellular calcium

383

mobilization and intracellular calcium-triggered synaptic vesicles are involved in

384

exocytosis (31). So we further treated the cells with intracellular calcium inhibitors to

385

explore the role of calcium in LPS-induced ATP release. As shown in Fig. 2C&D,

386

LPS-induced calcium mobilization was blocked by U73122 (inhibitor of PLC),

387

thapsigargin and caffeine (draining intracellular Ca2+ from the ER). As a consequence,

388

LPS-induced ATP release was also prevented by U73122, thapsigargin and caffeine

389

but not by CaCL2, CQ and BFA, suggesting the key role of calcium mobilization in

390

LPS-induced ATP release. Meanwhile, the in vivo data also showed NEM and U73122

391

negatively regulated the host defense against invading pathogens through disrupting

392

eATP release. Our data strengthen the correlation between TLRs and purinergic

393

signaling and also reveal novel evidence of the synergistic effect of pattern

394

recognition and danger signaling in fighting against invading pathogens.

395

Bacterial peritonitis is a serious and fatal complication due to the sustained

396

impairment of the immune system. Empirical therapy for peritonitis is mainly through

397

different antibiotics such as cefoxitin and cefotecan to cover gram positives, gram

398

negatives, and anaerobes. Thus, it will go a long way to reducing the abuse of

399

antibiotics if we can eliminate invading pathogens through the innate immune system. 19

TLR triggered ATP release enhances antibacterial immunity

400

Different kinds of immune cells have been found to be involved in the host defense

401

against infection, among which macrophages could be the most important.

402

Macrophages fulfill a series immune functions, including phagocytosis, antigen

403

presentation, and immunomodulation through cytokine and chemokine production

404

(32). So we treated macrophages with ATP to investigate its influence on

405

macrophage-mediated immune responses. In this study, not only could ATP be

406

released from macrophages, but the released ATP could also eliminate invading

407

bacteria and protect mice from bacteria-induced peritonitis (Fig. 3A&B). As shown in

408

Fig. 4&5, ATP exerted a protective effect during bacterial infection mainly through

409

increasing IL-1β and CCL-2 and promoting the phagocytosis of bacteria. These

410

findings provide solid evidence that eATP could serve as a positive regulator in

411

macrophage-mediated innate immunity.

412

As a natural ligand for all P2X and P2Y1, P2Y2, P2Y4, and P2Y11 receptors, ATP

413

could bind to these receptors and transduce intracellular signals in order to regulate

414

cell function (3). Almost all ATP-associated receptors are expressed in RAW 264.7

415

cells and PMs; among these, P2X7 and P2Y2 were highly expressed, implying a

416

potential role of ATP in regulating immune responses though P2 receptors (Fig. 4A).

417

Then we treated cells with the broad spectrum P2 receptor inhibitors PPADS and

418

Suramin to illustrate the role of P2X and P2Y in ATP- mediated immune regulation.

419

As shown in Fig. 4G&H, ATP induced CCL-2 and IL1-β were both blocked by

420

PPADAS, Suramin and apyrase, which further confirmed the key role of ATP in

421

cytokine and chemokine production and that this type of function is mediated mainly 20

TLR triggered ATP release enhances antibacterial immunity

422

through activating P2X and P2Y receptors. Similar data were also observed in the

423

phagocytosis assay. Furthermore, if we pretreated the mice with PPADAS and

424

Suramin, the ATP-mediated protection against bacterial peritonitis was also blocked.

425

Taken together, our study reveals an internal relationship between danger signals and

426

TLR signaling in innate immune responses, which suggests a potential therapeutic

427

significance of the ATP-associated signaling pathway in preventing and controlling

428

bacterial diseases.

429

21

TLR triggered ATP release enhances antibacterial immunity

430

Acknowledgments

431

Grant support: This work was supported by National Basic Research Program of

432

China [2012CB910401]; National Natural Science Foundation of China [81272369,

433

81172816]; Doctoral Fund of Ministry of Education of China [20130076110013];

434

Fundamental Research Funds for the Central Universities; Science and Technology

435

Commission

436

12XD1406100].

of

Shanghai

Municipality

[11DZ2260300,

11JC1414302,

437 438

Conflict of Interest

439

We declare that we have no conflict of interest.

440

22

TLR triggered ATP release enhances antibacterial immunity

441

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Figure legends

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Figure 1. Characterization of ATP release during bacterial infection. A. E. coli

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0111:B4 (2 × 108 CFU/ml) was injected into the mouse abdominal cavity. Then eATP

526

was detected using a luciferin-luciferase assay at different time points, and ATP was

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calculated according to a standard curve. B. RAW 264.7 cells, BMMs and PMs were

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stimulated with 100 ng/ml LPS at the times indicated, and then cultured cell

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supernatants were collected at the indicated times and subjected to ATP measurement.

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C. RAW 264.7 cells were treated with 100 ng/ml LPS or Panm3CSK4 for 15 minutes

531

to detect the release of ATP. Data are presented as means ± SEMs (n = 3, *p

Toll-like receptor-triggered calcium mobilization protects mice against bacterial infection through extracellular ATP release.

Extracellular ATP (eATP), released as a "danger signal" by injured or stressed cells, plays an important role in the regulation of immune responses, b...
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