1

Culture filtrate protein 10 kDa (CFP-10) from Mycobacterium tuberculosis selectively 1

activates human neutrophils through a pertussis toxin-sensitive chemotactic receptor 2

3

Running title: Neutrophils recognize M. tuberculosis CFP-10 4

5

Amanda Welin1#

, Halla Björnsdottir1, Malene Winther

1, Karin Christenson

1, Tudor Oprea

1,2, 6

Anna Karlsson1, Huamei Forsman

1, Claes Dahlgren

1, Johan Bylund

1,3 7

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1 The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, 10

Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden 11

2 Division of Biocomputing, Department of Molecular Biology, University of New Mexico 12

Health Sciences Center, Albuquerque, NM, USA 13

3 Department of Oral Microbiology and Immunology, Sahlgrenska Academy at University of 14

Gothenburg, Gothenburg, Sweden

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16

#Corresponding author e-mail address: amanda.welin@gu.se 17

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19

20

21

22

23

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IAI Accepts, published online ahead of print on 20 October 2014Infect. Immun. doi:10.1128/IAI.02493-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

2

Abstract 25

Upon infection with Mycobacterium tuberculosis (Mtb), neutrophils are massively recruited to 26

the lungs, but the role of these cells in combating the infection is poorly understood. Through a 27

type VII secretion system, Mtb releases a heterodimeric protein complex that is essential for 28

virulence, containing 6 kDa early secreted antigenic target (ESAT-6) and 10 kDa culture filtrate 29

protein (CFP-10). Whereas the ESAT-6 component possesses multiple virulence-related 30

activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been 31

described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex 32

induces a transient release of Ca2+

from intracellular stores in human neutrophils. Surprisingly, 33

CFP-10 rather than ESAT-6 was responsible for triggering the Ca2+

response, in a pertussis toxin-34

sensitive manner, suggesting the involvement of a G-protein coupled receptor. In line with this, 35

the response was accompanied by neutrophil chemotaxis and activation of the superoxide-36

producing NADPH-oxidase. Neutrophils were unique among leukocytes to respond to CFP-10, 37

as monocytes and lymphocytes failed to produce a Ca2+

signal upon stimulation with the Mtb 38

protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation 39

during Mtb infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 40

complex, beyond the previously described chaperone function. 41

42

43

44

45

3

Introduction 46

Human tuberculosis is caused by the bacterium Mycobacterium tuberculosis (Mtb), which enters 47

its host through the airways and can establish infection in the lungs. Inhalation of Mtb-containing 48

aerosols leads to one of three outcomes – clearance of the infection by the innate immune system, 49

establishment of a latent disease state where viable bacteria are contained within granulomas by 50

the combined action of the innate and adaptive immune systems, or development of active 51

tuberculosis (1, 2). The importance of innate immunity in overcoming an Mtb infection is 52

illustrated by the fact that up to 50% of exposed individuals are believed to clear the infection 53

without involvement of the adaptive immune system (3). The alveolar macrophage is considered 54

the main host cell of Mtb, and a lot of effort has been put into elucidating how bacteria manage to 55

survive inside these cells. 56

57

The short-lived and abundant neutrophils are the first leukocytes to respond to inflammatory 58

stimuli. Although it is clear that massive recruitment of neutrophils occurs upon Mtb infection as 59

a result of chemokine production at the site of infection, and that neutrophils phagocytose great 60

numbers of Mtb bacilli, much less is understood about their role in combating Mtb infection. 61

Conflicting data exist, but it is becoming clear that neutrophils can have both protective and 62

immunopathological effects in tuberculosis (4). 63

64

Mtb has a complex, lipid-rich cell wall, in which five known type VII secretion systems are 65

expressed, and these systems are optimized to effectively export material from the bacterial 66

cytoplasm to the extracellular space. The best studied of the secretion systems is the so called 6 67

kDa early secreted antigenic target (ESAT-6) secretion system 1 (ESX-1), which is encoded for 68

by the region of difference 1 (RD1) in the mycobacterial genome, and which is essential for Mtb 69

4

virulence. The ESX-1 is responsible for the transfer to the surrounding milieu of a heterodimeric 70

protein complex containing ESAT-6 (also known as EsxA) and a 10 kDa culture filtrate protein 71

(CFP-10, also known as EsxB or Mtb-specific antigen 10 (MTSA-10)) (5-9). The CFP-10 72

contains a C-terminal sequence that enables secretion of the complex from the bacterial 73

cytoplasm (10) and the complex is believed to dissociate under acidic conditions, e.g. inside a 74

phagolysosome (11). The ESAT-6:CFP-10 complex is an essential virulence factor of both Mtb 75

and M. marinum. It appears to have membrane-lysing activity (11-13), contributing to microbial 76

escape from the phagosome into the macrophage cytoplasm (13-15), and to induce host cell 77

necrosis and spread of bacteria to adjacent cells (12, 16). The complex has furthermore been 78

implicated in inflammasome activation (17) and inhibition of toll-like receptor (TLR) signaling 79

(18, 19) in monocytes, as well as in induction of IL-8 production in lung epithelial cells (20). The 80

CFP-10 part of the complex has been described as a chaperone protein for ESAT-6, responsible 81

for delivering its more biologically active binding partner to the site of action (11). 82

83

A recent study by Corleis et al. showed that upon phagocytosis by neutrophils, Mtb activates the 84

antimicrobial artillery of these cells, but then escapes by inducing neutrophil necrosis through an 85

RD1-dependent mechanism (21). Very little is, however, known about the interaction between 86

the RD1 gene product ESAT-6:CFP-10 and neutrophils. This prompted us to investigate the 87

direct interaction between ESAT-6:CFP-10 and human neutrophils. We found that neutrophils 88

were able to recognize the ESAT-6:CFP-10 complex and that CFP-10 rather than ESAT-6 was 89

the component recognized by the cells. CFP-10 stimulation of neutrophils resulted in a transient 90

release of Ca2+

from intracellular stores, accompanied by neutrophil chemotaxis and production 91

of reactive oxygen species (ROS). The CFP-10-induced Ca2+

and ROS responses were pertussis 92

toxin (PtX)-sensitive, suggesting the involvement of a G-protein coupled receptor (GPCR). 93

5

Neutrophils specifically recognized CFP-10, and no Ca2+

signal was induced in monocytes or 94

lymphocytes. Thus, this study shows that the CFP-10 component of ESAT-6:CFP-10 activates 95

human neutrophils, suggesting direct proinflammatory activity that may be of importance for the 96

Mtb – host interplay. 97

98

6

Materials and Methods 99

Cell separation 100

Peripheral blood neutrophils were separated from one day-old buffy coats from healthy blood 101

donors by dextran sedimentation and Ficoll-paque centrifugation according to Boyum (22, 23). 102

After separation and lysis of erythrocytes, the neutrophils were washed and resuspended in 103

Krebs-Ringer glucose (KRG) phosphate buffer supplemented with Ca2+

(1 mM) and stored on 104

melting ice until use. The purity of the preparation was typically around 95%. A whole leukocyte 105

preparation was obtained in the same way, but omitting the Ficoll-paque centrifugation. KRG 106

buffer with Ca2+

(1 mM) was used for all experiments. 107

108

ESAT-6 and CFP-10 109

Recombinant ESAT-6 and CFP-10, both purified from Escherichia coli, were purchased from 110

BBI Solutions or Fitzgerald Laboratories. Purity and identity were confirmed by the 111

manufacturers through Coomassie staining of protein samples on SDS-PAGE (ESAT-6 10.2 kDa, 112

CFP-10 11 kDa) and reactivity with serum from tuberculosis patients on Western blots. Further, 113

the preparations were analyzed by LC-MS/MS (The Proteomics Core Facility at Sahlgrenska 114

Academy, University of Gothenburg), confirming sequence identity. The endotoxin level was <1 115

EU/µg protein as determined by Limulus lysate assay (Clinical Microbiology, Sahlgrenska 116

University Hospital). ESAT-6:CFP-10 complex (molecular weight 21.2 kDa) was produced by 117

mixing equimolar concentrations of both proteins, as previously described (9, 19, 24). 118

119

Measurement of intracellular Ca2+

transients 120

Ca2+

mobilization from intracellular stores was measured using flow cytometry (25). Neutrophils 121

or mixed leukocytes (as indicated) were diluted in buffer supplemented with 1% fetal calf serum 122

7

to 8×106/ml and loaded with the Ca

2+ indicator dyes Fluo-3 (Molecular Probes; fluorescence 123

increases upon Ca2+

binding) and FuraRed (Molecular Probes; fluorescence decreases upon Ca2+

124

binding) at 37oC for 30 min. After two washing steps, the cells were diluted in buffer and stored 125

on ice until use. Before each run, the cells were equilibrated at 37oC for 3 min. Fluo-3/FuraRed 126

intensity was recorded continuously for 150 s using an Accuri C6 flow cytometer (BD Accuri). 127

After 30 s, stimuli (Mtb products or controls) were added as indicated. Formyl-methionyl-leucyl-128

phenylalanine (fMLF) (Sigma) and ionomycin (Sigma) were used as positive controls. 2.5 mM 129

EGTA (Sigma) was used to deplete the medium of Ca2+

. Analysis was performed using FlowJo 130

software (v. 7.6.5, TreeStar) and results are presented as the ratio between Fluo-3 and FuraRed 131

fluorescence intensities (normalized against the value at time=0), reflecting the relative cytosolic 132

Ca2+

concentration, over time. 133

134

Sytox green assay 135

In order to assess neutrophil plasma membrane integrity after addition of CFP-10, the membrane-136

impermeable Sytox green DNA dye (Molecular Probes) was used. 5×105 neutrophils per well 137

were seeded in black 96-well plates, diluted in buffer containing 2.5 µM Sytox green. CFP-10 or 138

the detergent Triton X-100 (TX100, Merck, 1%, positive control) was added to triplicate wells 139

and the plate was incubated at 37oC. Sytox green fluorescence was measured in a Mithras LB940 140

plate reader (Berthold Technologies) after 5, 30, and 120 min, and the median intensity value 141

from each triplicate was used. Results are presented as % of TX100 at each time point. 142

143

LDH assay 144

The effect of CFP-10 on neutrophil plasma membrane integrity was further assessed by the 145

lactate dehydrogenase (LDH) release assay (Cytotoxicity Detection Kit (LDH), Roche), as 146

8

described (26), after incubation of 5×105 neutrophils with CFP-10 or 1% TX100 at 37

oC for 2h. 147

Absorbance at 490 nm was measured, and results are presented as % of TX100. 148

149

Imaging flow cytometry 150

Visualization of neutrophil permeabilization by CFP-10 was performed by imaging flow 151

cytometry (ImageStreamX MkII, Amnis). 1×106 neutrophils were incubated with CFP-10 at 37

oC 152

for 30 min. During the last 5 min of incubation, the membrane-impermeable DNA dye Sytox 153

green (5 µM), which only stains permeabilized cells, and the membrane-permeable DNA dye 154

DRAQ5 (10 µM, Abcam), which stains all cells, were included. The cells were put on ice and 155

5000 images per sample were acquired in the imaging flow cytometer. Analysis was performed in 156

Ideas software (v. 6.0, Amnis). The proportion of Sytox green-positive cells was analyzed among 157

the in-focus, DRAQ5-positive, single cells. 158

159

Transwell chemotaxis assay 160

Neutrophil migration toward CFP-10 or fMLF (positive control) was analyzed by the ChemoTx 161

Chemotaxis System (NeuroProbe) according to the manufacturer’s instructions. Briefly, 30 µl 162

chemoattractant or buffer supplemented with 0.3% bovine serum albumin was placed in a well, a 163

filter with pore size 3 µm was applied, and neutrophils (3×104/sample diluted in buffer 164

supplemented with 0.3% bovine serum albumin) were added on top of the filter. Each 165

chemoattractant was run in triplicate and added below the filters. The plate was incubated at 37oC 166

for 90 min, after which the number of cells in the well (both attached and in the supernatant) was 167

assessed using microscopy and by quantification of LDH (as above) after lysis of the cells using 168

1% TX100. Median values were used, and results are expressed as % cell migration, calculated as 169

9

the % of the absorbance values obtained for a control well containing 3×104 cells placed below 170

the filter (100% cell control). 171

172

Measurement of NADPH-oxidase activity 173

NADPH-oxidase activity was determined using isoluminol-enhanced chemiluminescence (27, 174

28). Chemiluminescence was measured in a six-channel Biolumat LB 9505 (Berthold 175

Technologies), using disposable polypropylene tubes with a 360-µl reaction mixture containing 176

2×105 cells, 2×10

-5 M isoluminol and 2U horseradish peroxidase. The tubes were equilibrated in 177

the Biolumat for 10 min at 37°C, after which CFP-10 or fMLF (positive control) was added, and 178

light emission, corresponding to extracellularly released O2-, was recorded continuously. 179

Disruption of the actin cytoskeleton was performed by inclusion of 100 ng/ml latrunculin A 180

(Sigma) during equilibration. 181

182

Inhibition of G-protein coupled receptors 183

Pertussis toxin (PtX) was used to inhibit PtX-sensitive G-protein coupled receptors (GPCRs) 184

(29), and neutrophil activation in response to CFP-10 was measured in terms of Ca2+

mobilization 185

as above. During loading with Ca2+

indicator dyes, 500 ng/ml PtX (Sigma) was included in one 186

out of two vials, and after washing, PtX was again included in this vial for further incubation at 187

37oC, along with the control vial. As the time required to achieve G-protein inhibition by PtX 188

varies, inhibition was tested at 30 min intervals. This was done by stimulating PtX-treated or 189

untreated neutrophils with fMLF (GPCR-dependent) or phorbol myristate acetate (PMA, Sigma, 190

GPCR-independent) and recording O2- production as described above using chemiluminescence. 191

Once the response to fMLF was completely inhibited, but that to PMA remained intact, the Ca2+

192

10

experiment was performed on the treated cells. The total time of incubation with PtX was in the 193

range of 2.5 – 3 h. 194

195

Statistical analysis 196

The number of independent experiments (each performed on cells from an individual donor) is 197

stated in the figure legends, which also describe the statistical analyses performed (Graph Pad 198

Prism v. 6.01, GraphPad Software). Statistically significant differences are indicated in the 199

figures by * (p≤0.05), ** (p≤0.01), or *** (p≤0.001). Error bars in figures indicate the standard 200

deviation. 201

202

11

Results 203

Neutrophils recognize CFP-10 resulting in transient mobilization of intracellular Ca2+

204

As a transient rise in intracellular Ca2+

is an initial signal of fundamental importance in activation 205

of neutrophils and other leukocytes (30), we first determined the neutrophil response to the 206

ESAT-6:CFP-10 complex using the mobilization of cytosolic Ca2+

as a readout. Stimulation of 207

human neutrophils with the ESAT-6:CFP-10 complex gave rise to a Ca2+

signal comparable to 208

that induced by the formyl peptide receptor 1 (FPR1) agonist fMLF, used as a positive control 209

(Fig. 1A). Surprisingly, a similar molarity of CFP-10 alone induced a Ca2+

signal identical to that 210

evoked by the complex, while ESAT-6 alone induced no response (Fig. 1A). Thus, human 211

neutrophils recognized and responded with a Ca2+

transient to the ESAT-6:CFP-10 complex, and 212

the CFP-10 component was responsible for inducing this activity. The effect of CFP-10 was 213

concentration-dependent (Fig. 1B), with an activating concentration range similar to that used in 214

previous studies investigating binding of ESAT-6 or CFP-10 to cells and activating effects of 215

ESAT-6 (9, 19, 20). Having found that neutrophils recognize the CFP-10 component of ESAT-216

6:CFP-10, subsequent experiments was performed with CFP-10 alone. 217

218

The Ca2+

transient induced by CFP-10 occurs independently of plasma membrane disturbance 219

As it is well established that the ESAT-6:CFP-10 complex causes necrosis of macrophages (11-220

16), we next investigated the effect of CFP-10 on neutrophil viability. A plate-based assay 221

employing the impermeable DNA dye Sytox green showed that a breach of plasma membrane 222

integrity did not occur with the CFP-10 doses and time spans used to evoke mobilization of 223

intracellular Ca2+

. At higher concentrations or with prolonged exposure, a permeabilizing effect 224

of CFP-10 was noted (Fig. 2A) as seen in previous studies for pore formation by ESAT-6 in other 225

cell and membrane types (11, 13). A membrane-disrupting effect of CFP-10 at high concentration 226

12

was confirmed by increased levels of LDH, a commonly used marker of cell necrosis, in 227

neutrophil supernatants after prolonged incubation with CFP-10 at high concentration (Fig. 2B). 228

229

Importantly, CFP-10-induced membrane disruption did not cause cellular disintegration or 230

rupture, as shown by incubation with CFP-10 at high concentration and analysis by imaging flow 231

cytometry. Upon incubation with CFP-10 for 30 min, a significant proportion of the cells were 232

permeabilized (i.e. positive for Sytox green), but they were not disintegrated (Fig, 2C). The 233

results suggest pore formation rather than a detergent-like effect by high concentrations of CFP-234

10. 235

236

Plasma membrane permeabilization causes influx of ions and other small molecules from the 237

extracellular medium, resulting in increased cytosolic Ca2+

independently of receptor signaling. 238

To rule out the possibility that the Ca2+

signal induced by CFP-10 was due to such unspecific 239

leakage of Ca2+

, the Ca2+

chelator EGTA was added to the cells prior to stimulation. At the 240

concentration used (2.5 mM), EGTA chelates Ca2+

in the extracellular medium but does not 241

affect intracellular Ca2+

levels ((31) and data not shown). The presence of EGTA did not affect 242

the signal induced by CFP-10 (Fig. 2D). Thus, the cytosolic Ca2+

transient induced by CFP-10 243

was a true Ca2+

signal originating from an emptying of the intracellular storage organelles, a 244

phenomenon typical for neutrophils activated by an agonist binding to a chemoattractant receptor 245

(30, 31). 246

247

CFP-10 functions as a neutrophil chemoattractant and activates the superoxide-producing 248

NAPDH-oxidase 249

13

Having found that CFP-10 activates neutrophils, we next investigated the functional outcomes of 250

the interaction, focusing on neutrophil chemotaxis and production of ROS. The CFP-10 251

significantly induced cell migration in a concentration-dependent manner, as evidenced by a 252

plate-based trans-well system where cells were allowed to migrate across a filter towards CFP-253

10, or fMLF as a positive control (Fig. 3A). These results indicate that CFP-10 is indeed 254

chemotactic for human neutrophils. 255

256

Neutrophil activation by chemoattractants often results in activation of the electron transporting 257

NADPH-oxidase, and thus the production and release of ROS. The CFP-10 did not induce release 258

of extracellular superoxide (O2-) from unperturbed peripheral blood neutrophils (Fig. 3B). 259

However, many agonists require priming of the cells through degranulation and increased surface 260

expression of the corresponding receptor, or uncoupling from the actin cytoskeleton, to induce 261

ROS production (32). Latrunculin A interferes with the polymerization of actin (32) and 262

facilitates neutrophil secretion (33). When the cytoskeleton was disrupted through the addition of 263

latrunculin A to the cells prior to stimulation, CFP-10 triggered substantial ROS production (Fig. 264

3B), and the response to fMLF (positive control) was also enhanced (Fig. 3B). Taken together, 265

the functional outcomes of CFP-10-induced neutrophil activation include chemotaxis and ROS 266

production, pointing towards a proinflammatory response of neutrophils to the mycobacterial 267

protein. 268

269

CFP-10 induces neutrophil activation through a pertussis toxin-sensitive G-protein coupled 270

receptor specific for this cell type 271

Having found indications that CFP-10 interacts with a neutrophil chemoattractant receptor, we 272

next characterized the receptor that is responsible for the CFP-10-induced signal. The generation 273

14

of Ca2+

signals in neutrophils may be achieved through activation of numerous different 274

receptors, including members of the GPCR family of receptors which transmit their signals by 275

activating heterotrimeric G-proteins, usually of the Gi/o family (34). These G-proteins have in 276

common that they are sensitive to PtX, a Bordetella pertussis toxin which ADP-ribosylates the 277

Gα subunit of the G-protein, blocking receptor interactions and thus preventing activation (35). In 278

order to determine whether CFP-10 induces Ca2+

transients by binding to such a GPCR, 279

neutrophils were treated with PtX before stimulation with CFP-10 and measurement of the Ca2+

280

response. After PtX pre-treatment, the CFP-10-triggered Ca2+

response in neutrophils was 281

completely abolished (Fig. 4), demonstrating that CFP-10 utilizes a PtX-sensitive GPCR to 282

induce this response. The PtX treatment also blocked the Ca2+

signal induced by fMLF, which 283

acts through the PtX-sensitive GPCR FPR1, while the signal induced by the Ca2+

ionophore 284

ionomycin was not affected (Fig. 4). The PtX-treated neutrophils were fully able to produce ROS 285

in response to PMA stimulation (data not shown), affirming that the cells were viable and 286

functional also after PtX treatment. Moreover, the NADPH-oxidase activation induced by CFP-287

10 in latrunculin A-treated cells was likewise sensitive to PtX (Fig. S1), and thus mediated 288

through a PtX-sensitive GPCR. 289

290

To further characterize the unknown CFP-10 receptor, we tested several well-characterized 291

antagonists/inhibitors for a range of neutrophil GPCRs including FPR1, formyl peptide receptor 2 292

(FPR2), platelet activating factor (PAF) receptor, C5a receptor, P2Y2 receptor, and CXCR2. 293

However, none of the antagonists inhibited the CFP-10-induced Ca2+

signal (Fig. S2), indicating 294

that none of these GPCRs is the CFP-10 receptor. Instead, the cell specificity/selectivity of the 295

response was investigated by stimulating a mixed leukocyte population with CFP-10. The Ca2+

296

measurement technique was combined with immunofluorescence staining of the surface marker 297

15

CD45, differentially expressed on monocytes, lymphocytes, and neutrophils (36) (Fig. 5A). As 298

shown in Fig. 5A, CFP-10 induced Ca2+

mobilization only in neutrophils, but not in lymphocytes 299

or monocytes, demonstrating that the recognition of CFP-10 in this regard was neutrophil-300

specific. Addition of the Ca2+

ionophore ionomycin to the cells gave rise to an intracellular rise in 301

Ca2+

in all three cell types, showing that loading with Ca2+

indicators was effective for all 302

leukocyte subsets. As expected, stimulation with the FPR1 agonist fMLF yielded a Ca2+

signal in 303

monocytes and neutrophils, both of which express FPR1 (37), but not in lymphocytes. The 304

neutrophil-specificity of the CFP-10 response was further supported by experiments with the 305

human promyelocytic leukemia (HL-60) cell line that can be in vitro differentiated into either 306

neutrophil-like or monocyte-like cells (38), respectively. The CFP-10 induced a Ca2+

response in 307

neutrophil-differentiated, but not in monocyte-differentiated HL-60 cells (Fig. S3). Collectively, 308

these data indicate that neutrophils are unique among leukocytes in their ability to recognize and 309

respond to CFP-10 with a Ca2+

transient. 310

311

312

16

Discussion 313

Surprisingly little is known about how Mtb interacts with host neutrophils, even though these 314

cells are recruited in great numbers to the lung upon initial infection (4) and are present in 315

tuberculous granulomas (39). However, a recent publication showed that an intact RD1 genomic 316

region is required for Mtb to be able to evade neutrophil defense mechanisms and survive within 317

neutrophils (21). We set out to investigate the direct interaction between primary human 318

neutrophils and ESAT-6:CFP-10, a heterodimeric protein complex formed spontaneously by the 319

co-transcribed RD1-encoded proteins ESAT-6 and CFP-10 (5-9). Several cytolytic and immune 320

evasion functions of ESAT-6 have previously been revealed (12-16, 19), while CFP-10 has been 321

described to act as a chaperone protein for ESAT-6, required for its secretion (10), but without 322

known biological effects of its own (11, 19, 20). The data presented herein show that neutrophils 323

specifically recognize CFP-10, responding by cellular activation, and thereby reveal that the 324

CFP-10 component of the ESAT-6:CFP-10 complex possesses direct biological activity. 325

326

The ESAT-6:CFP-10 complex as well as CFP-10 on its own, but not ESAT-6 alone, induced an 327

intracellular Ca2+

transient in neutrophils. This shows that CFP-10 rather than ESAT-6 was 328

responsible for the neutrophil Ca2+

response to ESAT-6:CFP-10. Endotoxin contamination was 329

evidently not the cause of this neutrophil activation, since lipopolysaccharide stimulation does 330

not trigger Ca2+

signals even at very high concentrations (data not shown). ESAT-6:CFP-10 has 331

previously been shown to bind to the surface of U937 monocyte-like cells through the long 332

flexible arm formed by the C-terminus of CFP-10, and the structure of CFP-10 has been stated to 333

be consistent with a receptor-interacting function (9), in accordance with our findings. A few 334

earlier studies have indicated a possible inhibitory effect of CFP-10 on dendritic cell maturation 335

(40) and downregulation of inflammatory macrophage responses (41, 42). Several other studies, 336

17

however, have found a lack of direct effect of CFP-10 on different cell types, while ESAT-6 has 337

been shown to have several effects. These include interactions with TLR2 on macrophages, 338

impairing signaling (19), and the induction of IL-8 production in epithelial cells through an 339

unknown receptor (20). None of these studies included neutrophils. 340

341

As a functional ESX-1 secretion system is required for inducing macrophage lysis and Mtb 342

spread to adjacent cells (12, 13, 15, 16), we tested whether CFP-10 could also induce plasma 343

membrane permeabilization in neutrophils. CFP-10 at high concentrations permeabilized 344

neutrophils, but the cells appeared relatively intact by imaging flow cytometry after 30 min of 345

incubation, pointing towards pore formation in the neutrophil plasma membrane. It has 346

previously been shown that recombinant ESAT-6 is able to form pores in erythrocytes and 347

macrophages, as well as destabilize liposomes, while recombinant CFP-10 did not display this 348

effect in these cell types (11, 13). However, neutrophils have not previously been investigated. 349

350

CFP-10 permeabilized neutrophils in a manner resembling the effect of the phenol-soluble 351

modulin (PSMα) peptides produced by Staphylococcus aureus, substances also capable of 352

activating neutrophils through FPR2 at lower concentrations (43). Clearly, FPR2 was not 353

responsible for the activating effects of CFP-10 (Fig. S2), and the cytolytic effects of CFP-10 as 354

well as the PSMα peptides were insensitive to PtX (data not shown), indicating that the activating 355

and cytolytic properties of these bacterial factors are mediated by different mechanisms. Further, 356

the PSMα peptides selectively permeabilize apoptotic over viable neutrophils (43), but this was 357

not the case for CFP-10 (data not shown). 358

359

18

To characterize the neutrophil response to CFP-10, the functional outcome of the interaction was 360

investigated, and it was established that neutrophils responded to CFP-10 with both chemotaxis 361

and NADPH-oxidase activation. We speculate that the chemotactic property of CFP-10 may play 362

a role during early infection with Mtb – serving to attract neutrophils to the site of infection. 363

Although several Mtb factors have been shown to activate neutrophils (44, 45), this is to our 364

knowledge the first report of an Mtb protein that possesses direct chemotactic effects on 365

neutrophils. A variety of factors are known to indirectly attract neutrophils, e.g. ESAT-6 which 366

has been found to induce production of the neutrophil chemoattractant IL-8 in epithelial cells 367

(20). Similar to all prokaryotes however, Mtb initiates protein synthesis with formylated 368

methionine residues (46) and thus N-formylated peptides released from these bacteria would be 369

suspected to attract neutrophils directly through the action of FPRs. 370

371

Chemotaxis was induced by CFP-10 in unperturbed neutrophils, while priming with latrunculin 372

A, which disrupts filamentous actin, was required for NADPH-oxidase activation. The actin 373

cytoskeleton is known to be central for termination of GPCR signaling and when actin 374

polymerization is inhibited GPCR signaling is typically prolonged as well as enhanced (47). The 375

weak stimulatory effect of the NADPH-oxidase in resting cells by CFP-10, but potent effect on 376

cells with a disrupted cytoskeleton is similar to the neutrophil response through several other 377

neutrophil chemoattractant GPCRs (32, 48). The CFP-10 receptor differs from the FPRs in that 378

NADPH-oxidase activation through the receptor requires prior disruption of the actin 379

cytoskeleton, an observation that is likely explained by different signaling pathways leading from 380

receptor binding to activation of the NADPH-oxidase, in addition to differences in receptor 381

coupling to the actin cytoskeleton (29, 32, 49). 382

383

19

To pinpoint the CFP-10 receptor, PtX-sensitivity was studied, demonstrating that PtX abolished 384

both the CFP-10-induced Ca2+

response and NADPH-oxidase activation. Ptx is a specific 385

inhibitor of the Gi/o heterotrimeric G-proteins linked to the neutrophil chemoattractant receptors 386

(35), and our results thus strongly point towards the involvement of a classical chemoattractant 387

GPCR in mediating these responses. Several well-known chemotactic neutrophil GPCRs were 388

tested (FPR1, FPR2, PAF receptor, C5a receptor, P2Y2, and CXCR2) and can be excluded from 389

the list of potential receptors. Further, pretreatment with ESAT-6 before stimulation with CFP-10 390

in the NADPH-oxidase activity assay did not affect the CFP-10 response (data not shown), 391

indicating that ESAT-6 does not interact with the same receptor on neutrophils. The identity of 392

the CFP-10 GPCR remains unknown. It is possible that several different receptors are engaged by 393

the protein, mediating different responses. 394

395

Finally, experiments using mixed leukocyte preparations demonstrated that neutrophils were 396

unique among leukocytes in their ability to recognize and respond to CFP-10 with a Ca2+

397

transient, as monocytes and lymphocytes failed to respond. This indicates that the CFP-10 398

receptor is either only present or only functional on neutrophils, and not on the other cell types. 399

Alternatively, monocytes and/or lymphocytes do recognize CFP-10, but the interaction does not 400

result in the release of Ca2+

from intracellular stores. The idea that neutrophils harbor a 401

chemotactic GPCR that is not expressed (or not functional) on monocytes, which are also of 402

myeloid lineage, is intriguing and to our knowledge no such GPCR has previously been 403

described. On the other hand, the GPCR formyl peptide receptor 3 (FPR3) is expressed in 404

monocytes, but not in neutrophils (50), making it conceivable that such a receptor exists. Also, 405

experiments using monocyte- and neutrophil-differentiated HL-60 cells, where only the latter 406

responded to CFP-10, support our findings. Our data demonstrate that CFP-10 can signal through 407

20

a neutrophil-specific chemotactic GPCR of yet unknown identity, and we speculate that 408

recognition of CFP-10 through this receptor could be of benefit for the host in terms of rapid 409

recruitment of neutrophils that can eradicate Mtb infection, but also possibly for the bacterium 410

which then gains access to potential host cells. 411

412

In conclusion, this study demonstrates a novel direct biological role for the CFP-10 component of 413

ESAT-6:CFP-10 in specifically attracting and activating neutrophils. Thus there is apparently 414

more to CFP-10 than its previously described role as a chaperone protein for ESAT-6. We 415

speculate that neutrophils may encounter ESAT-6:CFP-10 during initial Mtb infection, prior to 416

phagocytosis or upon lysis of an Mtb-infected macrophage. This report adds to the growing body 417

of data indicating an important role of the ESAT-6:CFP-10 complex in the Mtb-neutrophil 418

interplay. 419

420

21

Acknowledgements 421

The authors declare no conflicts of interest. 422

423

This work was supported by the Swedish Heart-Lung Foundation (20130442), Swedish Research 424

Council (2012-1905, 2011-3358), King Gustav V Memorial Foundation, Gothenburg Medical 425

Society, Ingabritt and Arne Lundberg Research Foundation, the Clas Groschinsky Foundation, 426

and the Swedish state under the LUA/ALF agreement. 427

428

429

430

22

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605

606

607

30

Figure legends 608

Figure 1. Cytosolic Ca2+

transients induced by CFP-10 in neutrophils. Neutrophils were 609

loaded with Fluo-3 and FuraRed, and the fluorescence intensity ratio of the two dyes, 610

corresponding to relative Ca2+

concentration, was analyzed continuously during 150 s using flow 611

cytometry. A) Relative background Ca2+

level was recorded for 30 s, after which stimuli were 612

added (indicated by arrows). Neutrophils were stimulated with ESAT-6:CFP-10 complex (20 613

µg/ml), CFP-10 (10 µg/ml), ESAT-6 (10 µg/ml), or fMLF (10-7

M) as a positive control. The 614

curves show the normalized ratios between Fluo-3 and FuraRed over time, and are representative 615

of ≥3 independent experiments. B) Relative peak levels of cytosolic Ca2+

in neutrophils 616

stimulated with CFP-10 at the indicated concentrations, or buffer. The results are presented as 617

peak normalized Fluo-3/FuraRed ratios, and depict the mean of 4-10 experiments. A line is 618

shown at the background value. Statistical analysis was performed using ordinary one-way 619

ANOVA and Dunnett’s post-hoc test, comparing each concentration to the control. AU, arbitrary 620

units. 621

622

Figure 2. Effects of CFP-10 on plasma membrane integrity. Neutrophils were incubated at 623

37oC with or without CFP-10 and plasma membrane integrity was analyzed. A) A Sytox green 624

plate-based assay was used to assess neutrophil integrity upon the addition of CFP-10 at the 625

indicated concentrations, buffer only, or 1% TX100 as a positive control for plasma membrane 626

permeabilization. The diagram shows mean Sytox green fluorescence (expressed as % of TX100) 627

after 5, 30, and 120 min incubation, from 3-4 independent experiments. Statistical analysis was 628

performed using ordinary one-way ANOVA and Dunnett’s post-hoc test, comparing each 629

treatment to the non-treated control. B) The release of LDH from neutrophils incubated for 2 h 630

with buffer or CFP-10 at 50 µg/ml was assessed, and the diagram shows the mean LDH release 631

31

(expressed as % of TX100) from 5 independent experiments. Statistical analysis was performed 632

using paired Student’s t test. C) Neutrophils treated with or without CFP-10 at 50 µg/ml for 30 633

min were stained with DRAQ5 (stains DNA in all cells) and Sytox green (stains DNA only in 634

permeabilized cells), and analyzed by imaging flow cytometry. The images show brightfield 635

(BF), DRAQ5 (red) and Sytox green (green) images from 3 representative intact cells treated 636

with buffer, and 3 permeabilized cells treated with CFP-10. The diagram shows mean % 637

permeabilized (Sytox green-positive) cells from 3 independent experiments. Statistical analysis 638

was performed using paired Student’s t test. The scale bars represent 7 µm. D) Fluo-3 and 639

FuraRed fluorescence was analyzed in neutrophils during 150 s using flow cytometry in the 640

presence or absence of the Ca2+

chelator EGTA (2.5 mM). Stimulus (10 µg/ml CFP-10) was 641

added after 30 s (indicated by arrows). Normalized Fluo-3/Fura-Red fluorescence ratios, 642

representing relative Ca2+

levels, are shown and traces are representative of 3 independent 643

experiments. AU, arbitrary units. 644

645

Figure 3. Neutrophil chemotaxis and NADPH-oxidase activation induced by CFP-10. A) 646

Neutrophils were allowed to migrate across a membrane into a well containing buffer as a 647

negative control, fMLF (10-8

M) as a positive control, or CFP-10 at the indicated concentrations. 648

After 90 min, chemotaxis was evaluated by lysing the cells in the well and measuring LDH in the 649

lysates, corresponding to the number of cells present. The results are presented as mean % cell 650

migration (i.e. % of the 100% cell control). The dose-response curve shows median values from 651

one representative experiment, and the bar graph shows the mean of 7 independent experiments. 652

Statistical analysis was performed using paired Student’s t-test. Images show wells from each 653

treatment in one representative experiment. The scale bars represent 100 µm. B) NADPH-oxidase 654

activity was assessed in resting neutrophils or neutrophils pre-treated with latrunculin A (100 655

32

ng/ml) for 10 min. The cells were equilibrated at 37oC in a luminometer before addition of 656

stimuli (arrows) and recording of chemiluminescence over time (expressed in mega counts per 657

minute, Mcpm). The diagrams show extracellular O2- production over time in cells stimulated 658

with 10 µg/ml CFP-10 or 10-7

M fMLF (positive control). One representative experiment out of 3 659

is shown. 660

661

Figure 4. PtX sensitivity of the CFP-10-induced Ca2+

signal. Neutrophils were loaded with 662

Fluo-3 and FuraRed and treated with PtX to inhibit G-proteins, or left untreated. The 663

fluorescence ratio was analyzed continuously during 150 s using flow cytometry. A) Background 664

Fluo-3/FuraRed intensity was recorded for 30 s, after which 10 µg/ml CFP-10 (top panel), 10-8

M 665

fMLF (middle panel), or 10-8

M ionomycin (bottom panel) were added (indicated by arrows), at 666

the specified concentrations. The diagrams show the normalized ratios between Fluo-3 and 667

FuraRed fluorescence intensities, reflecting relative cytosolic Ca2+

levels, over time. B) The bar 668

graph shows the mean peak normalized fluorescence ratios between Fluo-3 and FuraRed, 669

reflecting relative peak cytosolic Ca2+

concentration, after pre-treatment and stimulation as 670

indicated, from 4 independent experiments. A line is shown at the background value. Statistical 671

analysis was performed using ordinary one-way ANOVA and Sidak’s post-hoc test, comparing 672

each stimulus with and without PtX treatment. AU, arbitrary units. 673

674

Figure 5. Neutrophil specificity of the CFP-10 Ca2+

response. Mixed leukocyte populations 675

were loaded with Fluo-3 and FuraRed, and then stained using anti-CD45 antibody to allow 676

discrimination of leukocyte subsets during Ca2+

measurements by flow cytometry. The ratio of 677

the fluorescence intensities from the two Ca2+

indicator dyes was recorded continuously during 678

150 s in lymphocytes, monocytes, and neutrophils. Background Fluo-3/FuraRed intensity was 679

33

recorded for 30 s, after which stimuli were added (indicated by arrows). The stimuli were 10 680

µg/ml CFP-10, 10-7

M fMLF, or 10-8

M ionomycin, as indicated. The dot plot shows CD45 681

intensity versus side scatter, and the gates used for the different cell types. The diagrams show 682

the normalized ratios between Fluo-3 and FuraRed fluorescence intensities, reflecting relative 683

cytosolic Ca2+

level, over time in the indicated cell populations, and are representative of 3 684

independent experiments. AU, arbitrary units. 685