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
2
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,
7
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
14
School of Life Sciences, East China Normal University, 500 Dongchuan Road,
15
Shanghai 200241, China; Tel: +86-21-54341039; Fax: +86-21-54344922;
16
E-mail:
[email protected] 17
Bing Du, Institute of Biomedical Sciences and School of Life Sciences, East
18
China Normal University, 500 Dongchuan Road, Shanghai 200241, China; Tel:
19
+86-21-24206964; Fax: +86-21-54344922; E-mail:
[email protected] 20
1
TLR triggered ATP release enhances antibacterial immunity
21
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
48
Introduction
49
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
74
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
77
recognize pathogen-associated molecular patterns (PAMPs) by germline-encoded
78
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
92
novel approach to better understand immune regulation.
93
In this study, we demonstrated that ATP was released from immune cells during
94
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,
97
we also provide evidence that ATP has a non-redundant role in promoting host
98
survival from E. coli induced peritonitis. TLR signaling not only can be involved in
99
inflammation responses through the typical MAPK pathway but can also regulate
100
innate immune responses via increasing extracellular “Danger signals” such as eATP.
101
Taken together, our study has shown a novel role of TLR-triggered calcium
102
mobilization in the upregulation of innate immune responses against bacterial
103
infection through extracellular ATP release.
104 105
Materials and Methods
106
Animals
107
For peritoneal macrophage (PM) and bone marrow macrophage (BMM) isolation, as
108
well as the peritonitis mouse model, female 6–8 week old C57BL/6 mice were
109
purchased from the Shanghai Laboratory Animal Company (Shanghai, China). All
110
mice used in these experiments were housed under pathogen-free conditions and were
111
maintained in accordance with institutional guidelines. All experimental protocols
112
were approved by the Animal Investigation Committee of East China Normal
113
University. 6
TLR triggered ATP release enhances antibacterial immunity
114
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
136
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
180
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
202
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
268
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
378
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
525
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
527
calculated according to a standard curve. B. RAW 264.7 cells, BMMs and PMs were
528
stimulated with 100 ng/ml LPS at the times indicated, and then cultured cell
529
supernatants were collected at the indicated times and subjected to ATP measurement.
530
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