Nieminen MT et al. Anti-inflammatory effect of HICA in vivo

D,L-2-hydroxyisocaproic acid (HICA) attenuates inflammatory responses in a 1

murine Candida albicans biofilm model 2

Nieminen MT1,2,4, Hernandez M5, Novak-Frazer L4, Kuula H2, Ramage G6, Bowyer 3

P4, Warn P4, Sorsa T2,3, Rautemaa R4 4

5

1. Research Unit on Acetaldehyde and Cancer, University of Helsinki, Finland 6

2. Departments of Periodontology and Oral and Maxillofacial Diseases, Helsinki 7

University Central Hospital, University of Helsinki, Helsinki, Finland 8

3. Division of Periodontology, Department of Dental Medicine, Karolinska 9

Institutet, Huddinge, Sweden 10

4. The University of Manchester, Institute of Inflammation and Repair, 11

Manchester Academic Health Science Centre, University Hospital of South 12

Manchester, Wythenshawe Hospital, Manchester, United Kingdom 13

5. Department of Oral Pathology and Medicine, and Laboratory of Periodontal 14

Biology, Faculty of Dentistry, Universidad de Chile, Chile 15

6. Infection and Immunity Research Group, Glasgow Dental School and 16

Hospital, School of Medicine, College of Medicine, Veterinary and Life 17

Sciences, Faculty of Medicine, University of Glasgow, Glasgow, United 18

Kingdom 19

20

Key words: Candida, biofilm, murine, 2-hydroxyisocaproic acid 21

Short title: Anti-inflammatory effect of HICA in vivo 22

23

Corresponding Author: 24

Dr. Riina Rautemaa-Richardson 25

Education & Research Centre, 2nd Floor 26

Wythenshawe Hospital, Southmoor Road 27

Manchester 28

M23 9LT 29

UK 30

email: riina.rautemaa@manchester.ac.uk 31

Fax: +44 161 291 5941 32

33

CVI Accepts, published online ahead of print on 2 July 2014Clin. Vaccine Immunol. doi:10.1128/CVI.00339-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Nieminen MT et al. Anti-inflammatory effect of HICA in vivo 1

Abstract 34

35

Chronic biofilm infections are often accompanied by a chronic inflammatory 36

response thus leading to impaired healing and increased, irreversible damage to 37

host tissues. Biofilm formation is a major virulence factor for Candida albicans and a 38

challenge for treatment. Most current antifungals have proven ineffective in 39

eradicating infections attributed to biofilms. The biofilm structure protects Candida 40

against antifungals and provides a way to evade host immune systems. This leads to 41

a very distinct inflammatory response compared to planktonic counterparts. 42

Previously, we have shown the superior efficacy of D,L-2-hydroxyisocaproic acid 43

(HICA) against various bacteria and fungi. However, the immunomodulatory 44

properties of HICA have not been studied. Our aim was to investigate the potential 45

anti-inflammatory response to HICA in vivo. We hypothesized that HICA reduces the 46

levels of immune mediators and attenuates the inflammatory response. In a murine 47

model a robust biofilm was formed for five days in a diffusion chamber implanted 48

underneath the mouse skin. The biofilm was treated for 12h with HICA while 49

caspofungin and PBS were used as controls. The pathophysiology and 50

immunoexpression in the tissues surrounding the chamber was determined by 51

immunohistochemistry. Histopathological examination showed an attenuated 52

inflammatory response together with reduced expression of matrix metalloproteinase 53

9 (MMP-9) and myeloperoxidase (MPO) compared to chambers containing 54

caspofungin and PBS. Interestingly, the expression of Del-1, an antagonist of 55

neutrophil extravasation, increased after treatment with HICA. Considering its anti-56

inflammatory and antimicrobial activity, HICA could provide an enormous therapeutic 57

potential in the treatment of chronic biofilm infections and inflammation, such as 58

chronic wounds. 59

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Nieminen MT et al. Anti-inflammatory effect of HICA in vivo 2

Introduction 60

61

Approximately 65% of human infections are biofilm-related (1). A residing biofilm 62

infection often causes aggravated inflammation in host tissues thus leading to a 63

chronic inflammatory status (2). Chronic inflammatory responses complicate healing 64

and cause increased and irreversible damage to host tissues which are 65

characteristic of chronic wounds and periodontitis (3, 4). 66

67

Candida albicans is an opportunistic fungal pathogen and causes both superficial 68

and systemic infections in humans (5). Infection arises when the yeast is able to 69

overcome the host immune response and this interplay is regulated by pro- and anti-70

inflammatory mediators. The carbohydrate extracellular matrix of biofilms provides a 71

very distinctive and protective niche for yeast cells to grow within the host and the 72

cells often show an altered phenotype and antifungal resistance profile compared to 73

planktonic counterparts (6, 7). Cells embedded within biofilms are able to evade host 74

immune cells since the cell surface structures are masked (1). Very few studies have 75

assessed the inflammatory response induced by C. albicans biofilm in vivo (8-10). 76

77

The management of Candida infections is challenging due to poor efficacy, patient 78

compliance and numerous side effects and interactions of commonly used 79

antifungals (5). The most promising anti-biofilm activity has been observed with the 80

echinocandin class antifungals, which are non-competitive inhibitors of (1,3)-β-D-81

glucan synthase, an essential enzyme in fungal cell wall synthesis and integrity (11, 82

12). Caspofungin is the most extensively used echinocandin, especially in the 83

treatment of invasive candidiasis (13, 14). Recently, more attention has been drawn 84

to the immunopharmacological properties of antifungals, for example, echinocandins, 85

whose mode of action has been shown to be dependent on these properties (15, 86

16). 87

88

The superior antifungal activity of a leucine derivative D,L-2-hydroxyisocaproic acid 89

(HICA) against C. albicans biofilms, compared with caspofungin has been 90

demonstrated (17). The efficacy of HICA against a spectrum of planktonically grown 91

bacteria and fungi has been reported (18, 19). HICA is a α-hydroxy amino acid 92

produced during Lactobacillus fermentation and also found in human tissues (20, 93

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Nieminen MT et al. Anti-inflammatory effect of HICA in vivo 3

21). It has been used by professional athletes for muscle recovery and for veterinary 94

purposes such as an animal feed thus demonstrating its biocompatibility and safety 95

profile (21, 22). Multiple studies have described the potential anti-inflammatory 96

properties of Lactobacilli and their metabolic products (23, 24). 97

98

The aim of this study was to determine the potential anti-inflammatory effects of 99

HICA in a C. albicans biofilm murine chamber model. To elucidate changes in the 100

local inflammatory response, we used immunohistochemistry to detect the 101

expression of immune proteases and other inflammatory mediators belonging to the 102

oxidative tissue destructive cascade, which is known to play major role in 103

inflammatory diseases such as periodontitis (3). The core of this cascade is 104

characterised by matrix metalloproteinase (MMP) activation by polymorphonuclear 105

neutrophil (PMN) cells secreted myeloperoxidase (MPO). Our hypothesis was that 106

HICA attenuates the anti-inflammatory response by altering expression of tissue 107

proteases and endogenous pro-inflammatory mediators. 108

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Materials and methods 109

110

Ethics statement 111

All animals were handled in strict accordance with good animal practice as defined in 112

the United Kingdom Animals (Scientific Procedures) Act. Animal experiments were 113

conducted under the ethically reviewed license authorized by the Secretary of State 114

to the University of Manchester, Manchester, UK (license no. PPL 40/3101). 115

116

Murine chamber model 117

A previously published chamber model was adapted for this study (25). The biofilm 118

chamber was structurally based on a diffusion chamber kit (Millipore, Watford, UK) 119

comprising of a semipermeable Durapore® membrane with a pore size of 0.45 µm 120

fixed to a Plexiglas® ring. A non-permeable silicon sheet was fixed to the opposite 121

side to face the semipermeable membrane and to close the chamber, and the 122

chambers were sterilized prior to use. A total of 24 male CD1 mice weighing 21-24 g 123

were used but one mouse was lost due to bleeding in surgery. The dorsal flank of 124

each mouse was shaved and a 2 cm incision was made. The diffusion chamber was 125

implanted subcutaneously, so that the semipermeable membrane faced the dorsal 126

muscles and the non-permeable silicon sheet the skin. The wound was closed with 127

non-absorbable braided silk sutures (Ethicon, NJ, USA) and meloxicam (3mg/kg) 128

was administered intraperitoneally daily for 3 days post-surgery. A week after 129

surgery, the chambers were injected percutaneously with 100 µL of C. albicans 130

SC5314 inoculum (106 CFU/mouse) under isoflurane anesthesia. The inoculum was 131

mixed thoroughly before injection and the inoculum concentration was checked using 132

dilution plating. The mice were left to recover for 5 days allowing robust C. albicans 133

biofilms to be formed formed inside the chambers. Then 100 µL of 5% (w/v) HICA, 134

10 mg/L caspofungin or PBS was injected percutaneously into the chambers. The 135

mice were euthanized 12 h post-treatment with an overdose of isoflurane. The 136

chambers were collected and the biofilms detached and weighed. Tissues around 137

the chambers were dissected and fixed and stored in 10% formaldehyde until 138

analyses. 139

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Study design 140

A total of 24 mice were used in this study. Diffusion chambers (Millipore, Watford, 141

UK) with a semipermeable membrane facing the tissues were implanted 142

subcutaneously in the dorsal flank of each mouse and the animals were allowed to 143

recover for seven days. The mice were divided into two main groups: a biofilm 144

(n=15) and a non-infected, non-biofilm (n=8) group. The chambers in the biofilm 145

group were infected with C. albicans and a robust biofilm was established over five 146

days. Biofilms were treated for 12h with HICA (n=8) and caspofungin (n=3) or PBS 147

(n=4) were used as control treatments. The non-biofilm chambers were treated 148

similarly with HICA (n=2), caspofungin (n=3) or PBS (n=3). The non-biofilm HICA 149

group was smaller due to a loss of one mouse in surgery (Final n=23). Mice were 150

euthanized post-treatment and chambers and surrounding subcutaneous tissue 151

sections were collected from each mouse. Biofilms were detached from the 152

chambers and weighed. To analyze the changes in cellular and tissue structures and 153

the extent of inflammatory response, tissue sections were stained with hematoxylin-154

eosin and for matrix metalloproteinase -8, -9 (MMP-8, MMP-9), myeloperoxidase 155

(MPO), neutrophil elastase (NE), tumor necrosis factor -alpha (TNFα), interleukin 1 -156

beta (IL1β) and developmental endothelial locus 1 (Del-1) using the corresponding 157

antibodies. Stained sections were evaluated by light microscopy and the staining 158

intensity was semi-quantified and graded. 159

160

Strain and growth conditions 161

C. albicans SC 5314 was used in this study (26). The strain was stored at -80°C, 162

plated twice on Sabouraud dextrose agar (Melford, Suffolk, UK) and incubated at 163

37°C for 48h before use to check for viability and purity. A colony was suspended 164

into PBS, mixed well and the cells were washed twice before adjusting the inoculum 165

using a haemocytometer to correspond to 107 CFU/mL. Viable counts were verified 166

by dilution plating. Viability of biofilms was checked by culture after the treatments 167

with PBS, caspofungin or HICA at the end of the experiment. 168

169

Immunohistochemistry 170

Immunohistochemical staining was performed as described previously (27). Briefly, 171

tissue sections were embedded in paraffin. Paraffin-embedded specimens were 172

sectioned, deparaffinised, pretreated with 0.4% pepsin, and endogenous peroxidase 173

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activity was blocked with H2O2/methanol. Staining was performed using either 174

polyclonal Vectastain Elite rabbit or goat avidin-biotin enzyme complex (ABC) kits 175

(Vector Laboratories, Burlingame, CA, USA). Sections were blocked with goat or 176

rabbit normal serum in 2% bovine serum albumin and incubated with the following 177

polyclonal antibodies: rabbit MMP-8 (Santa-Cruz Biotechnology, Santa-Cruz, CA, 178

USA), goat MMP-9 (R&D Systems, Minneapolis, MN, USA), rabbit MPO (Hycult 179

Biotechnology, Uden, Netherlands), rabbit NE (Calbiochem, San-Diego, CA, USA), 180

goat IL1β (R&D Systems, Minneapolis, MN, USA), goat TNFα (R&D Systems, 181

Minneapolis, MN, USA) and rabbit Del-1 (Proteintech, Chicago, IL, USA). Control 182

sections were incubated with non-immune rabbit or goat serum. The inflammatory 183

markers were visualised using a biotinylated anti-rabbit or anti-goat secondary 184

antibody and avidin-biotin enzyme complex. 3-amino-9-ethyl-carbazole was used as 185

a chromogen and Mayer’s hematoxylin (Histolab Products AB, Frölunda, Sweden) as 186

counterstain. All sections were stained also with hematoxylin and eosin (H&E) for 187

routine histopathology. 188

189

Stained sections were evaluated under an Olympus BX61 light microscope and 190

representative images were taken using an Olympus DP50 camera and analysed by 191

AnalySIS-software (AnalySIS ver. 3.2, Soft Imaging System GmbH, Muenster, 192

Germany). Immunoreactivity of the tissue sections surrounding the chamber was 193

semiquantified for each protein antibody and graded based on the staining intensity 194

as - (no staining), + (mild), ++ (moderate) and +++ (strong). The results from the 195

semi-quantitative analysis were confirmed blindly by a second evaluator and a 196

trained pathologist examined the histopathology. Distributions in staining intensity 197

within groups were visualized using Graphpad Prism v.5.0 software (GraphPad 198

Software Inc., La Jolla, CA, USA). 199

200

Statistical analysis 201

The data was analysed using Graphpad Prism software ver. 5.0 (Graphpad Software 202

Inc.). The univariate analysis of variance (ANOVA) was used for comparisons 203

between biofilm weights. P-values of less than 0.05 were considered significant. 204

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Results 205

206

C. albicans biofilms 207

No significant differences were measured in the biofilm weights after treatment for 208

12h with HICA, caspofungin or PBS (p=ns). The lowest average weight (18.5±7.1 209

mg) was measured for biofilms treated with HICA whereas the highest mean weight 210

(20.8±7.9 mg) was measured for caspofungin treated biofilms. All biofilms were 211

viable after the treatments with PBS, caspofungin or HICA. 212

213

Histopathology 214

Upon histopathological examination (H&E staining), typical components of wound 215

healing and varying levels of inflammation were seen in all tissue samples. Debris 216

and fibrin together with varying amounts of polymorphonuclear neutrophils (PMNs) 217

were present in the tissues that had been resting against the semipermeable 218

membrane of the chamber. The underlying granulation tissue presented varying 219

degrees of mixed inflammatory infiltrate. The intensity of the inflammatory response 220

was different within and between groups. The degree of cellular density and edema 221

also varied. When biofilm and non-infected, non-biofilm groups were compared, 222

marked differences were seen. In all non-biofilm sections granulation tissue was a 223

thin layer and inflammation mainly composed of lymphocytes, plasma cells and 224

monocytes. The density was altogether moderate. Interestingly, the biofilm-HICA 225

group showed an inflammatory response similar to that seen in the non-biofilm 226

control sections, that is, a predominantly mononuclear cellular infiltrate moderate in 227

density. However, a PMN infiltrate was observed superficially compared to sections 228

in the non-biofilm groups. The two biofilm groups treated either with caspofungin or 229

PBS showed a thicker band of granulation tissue with a dense inflammatory cell 230

infiltrate, composed mostly of macrophages and PMN cells. In addition, inflammatory 231

foci in muscle and adipose layers and abscess formation varying in size were 232

frequently observed in the biofilm group, particularly in caspofungin and PBS 233

controls. 234

235

Semi-quantitative immunohistochemical analysis 236

Staining for MMP-8, MMP-9, MPO, NE, IL1β and TNFα predominantly localised in 237

the inflammatory cells in all groups. In the biofilm groups, the staining for MPO, 238

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MMP-8 and MMP-9 was less intense after HICA treatment compared to caspofungin 239

and PBS (Fig.1, Fig. 2A,B). The most distinct differences between the treatment 240

groups were observed in the staining intensities of MPO and MMP-9. However, no 241

marked differences were seen in MPO, MMP-8 or MMP-9 staining intensities 242

between the treated and untreated non- infected, non-biofilm controls (Fig. 1, Fig. 243

2A,B). In general, staining for MPO, MMP-8 and MMP-9 was stronger in the biofilm 244

group than in the non-biofilm group. The staining of NE was stronger in the biofilm 245

group than in the non-biofilm group, although less intense than for MPO (Fig. 1). 246

However, in contrast to MPO no marked differences could be seen between the 247

treatments. The expression of IL1β was also stronger in the biofilm group with 248

minimal differences between treatments (Fig. 1). In contrast, minimal TNFα staining 249

was seen in all treatments and groups (Fig. 1). 250

251

In the non-biofilm group, Del-1 was strongly expressed by endothelial cells in all 252

treatment groups (Fig. 1 and Fig. 2C). The staining was clearly less intense in the 253

untreated and caspofungin biofilm groups. However, in the HICA treated group 254

moderate to strong staining was seen in the endothelial cells adjacent to the 255

chambers. 256

257

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Discussion 258

259 This is the first study to address the impact of HICA on the inflammatory response to 260

infection in vivo. Less inflammation was observed in tissues surrounding the biofilm-261

infected diffusion chamber after treatment with HICA compared to caspofungin and 262

PBS. Histopathology showed a predominantly mononuclear cell profile and a less 263

prominent and less dense PMN infiltrate. A decrease in MPO expression was 264

observed after HICA treatment. This correlated with the decreased expression of 265

MMP-9 in tissue sections and indicates a reduced oxidative inflammatory burden as 266

a result of the MPO and MMP-9 cascade. Significant reductions between HICA and 267

controls with regard to MMP-8 and NE expression were not seen. 268

269

High expression of MMP-8, MMP-9, MPO and NE has been detected in chronic 270

inflammatory diseases and linked to loss of soft and hard tissues (3, 28). However, 271

numerous studies have described the anti-inflammatory effect of MMP-8 and shown 272

its role in wound healing (27, 29, 30). NE, secreted by PMN cells, plays an important 273

role in wound healing but prolonged and excessive levels can impair the healing 274

process as observed in chronic wounds (31). This underlines the importance of 275

homeostasis in the expression of immune mediators. 276

277

Tissue sections in the caspofungin and PBS biofilm groups showed characteristics of 278

chronic inflammation since abscess formation and inflammation in deeper tissue 279

layers were frequently observed. This correlated with the staining pattern 280

representing the expression (Fig. 1). In vivo studies have shown that caspofungin 281

exerts its immune-modulatory effects through the morphological changes in the 282

fungal cell wall structure as a result of increasing β-glucan exposure which leads to 283

an increased inflammatory response (32, 33). 284

285

In humans, HICA is a by-product of ketoisocaproic acid (KICA) in the leucine 286

pathway (34). Multiple studies have investigated the immune-modulatory role of 287

leucine and anti-catabolic and anti-inflammatory activities have been observed (35, 288

36). A study using a combination of herbs and leucine for the treatment of articular 289

diseases showed an induction of IL1β and strong down-regulation of MMP-9 (37), 290

similar to the effect seen in our study. To further support our hypothesis, multiple 291

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studies have shown the potential anti-inflammatory effect of Lactobacillus 292

metabolites (23, 24). This is relevant because the antimicrobial activity of HICA was 293

discovered in a mixture of Lactobacillus plantarum fermentation products (38, 39). 294

295

Interestingly, endothelial cell-secreted protein Del-1 showed stronger expression in 296

the HICA treated biofilm-group compared to post-treatment with either caspofungin 297

or PBS. The staining profile was similar to non-biofilm controls (Fig. 1, Fig 2C). Del-1 298

has been linked to inflammatory diseases such as periodontitis and Sjögren 299

Syndrome (40, 41). In addition to its role as an inhibitory agent against intercellular 300

adhesion molecule 1 (ICAM-1) -dependent neutrophil adherence to lymphocyte 301

function-associated antigen 1 (LFA-1) –integrin and extravasation, a recent study 302

described its inhibitory action against ICAM-1-dependent chemokine release from 303

neutrophils thus potentiating its regulatory role and further extends to inflammatory 304

circuitry (42). Our observations support the results by others and provide evidence of 305

a potential anti-inflammatory shield induced by HICA. 306

307

A single-dose of neither HICA nor caspofungin showed major inhibitory activity 308

against C. albicans biofilms in 12-hour incubation. This is in line with the results of 309

previous studies where no major antifungal activity against a fully mature biofilms 310

was observed in caspofungin lock therapy in vivo when similar short treatment times 311

were used (43). In our model, biofilms were left to form for five days before 312

treatment. The properties and structure of such biofilms have been shown to 313

correlate with mature (24 or 48h) in vitro biofilm (44). In our in vitro study, HICA was 314

highly active against mature biofilms after 24h treatment (17). The different 315

inflammatory response observed after HICA treatment could be affected by its 316

deteriorating effect on biofilm ultrastructure and fungal cells. 317

318

Our study is one of the few studies presenting the inflammatory response against a 319

fully mature C. albicans biofilms in vivo. In vitro co-culture study with C. albicans 320

biofilms and mononuclear cells showed a strikingly different pro- and anti-321

inflammatory cytokine profile compared to planktonic cells (45). IL-1β was 322

significantly up-regulated and in comparison TNFα was significantly down-regulated. 323

Multiple cell culture and in vivo studies have shown the similar down-regulation of 324

TNFα in bacterial biofilm infections (46-48). Our findings are in line with the previous 325

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Nieminen MT et al. Anti-inflammatory effect of HICA in vivo 11

studies and further support the view that biofilms induce a distinct immune response 326

(Fig. 1). Interestingly, a recent study showed that neutrophils can modulate the 327

inflammatory response by inhibiting TNFα and IL1β expression (49). 328

329

HICA can increase protein synthesis and improve muscle recovery after 330

immobilization-induced atrophy (50). The induction of protein synthesis was 331

interpreted to occur through activation of mTOR signaling. Interestingly, innate 332

inflammatory responses induced by bacteria, fungi, parasites and viruses have also 333

been shown to be regulated by the mTOR pathway (51, 52). In addition, protection 334

against mucosal damage during C. albicans infection is mediated through mTOR 335

activation (53). These findings providing evident clues for the potential action of 336

HICA to exert its anti-inflammatory and protective effects should be addressed in 337

future studies. 338

339

Biofilm infections are challenging to manage, especially in patients with a 340

compromised immune system. In addition to their efficacy against the microbial 341

pathogens, attention should also be aimed at the immune-modulatory activity of 342

antimicrobial agents. Considering the antimicrobial efficacy of HICA and its potential 343

anti-inflammatory activity, HICA could provide a huge therapeutic potential in the 344

treatment of chronic biofilm infections and inflammation, such as chronic wounds. 345

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Acknowledgements 346

347

This work was financially supported by the Biotechnology and Biological Sciences 348

Research Council, Engineering and Physical Sciences Research Council, EU 349

Framework 7, Finnish Dental Society Apollonia, Fondo Nacional de Desarrollo 350

Científico y Tecnológico, Gilead Sciences, GlaxoSmithKline, Helsinki University 351

Central Hospital Research Foundation, National Aspergillosis Centre UK, Orion 352

Research Foundation, Yrjö Jahnsson Foundation and the Wellcome Trust. 353

354

We thank Dr. Taina Tervahartiala for valuable advice regarding the technical and 355

analytical issues and Marjatta Kivekäs for her skilful technical assistance. 356

357

TS is one of the inventors of European Patent Office patent no. EP0871438B1: “Use 358

of alpha-hydroxy acids in the manufacture of a medicament for the treatment of 359

inflammation” (39). TS has not received any royalties regarding this patent. PW is 360

the chief scientific officer, director and shareholder of Euprotec Ltd which is a 361

contract research company and provides discovery services to multiple companies 362

that develop treatments and vaccines for infectious diseases. 363

364

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Figure legends 543

544

FIG 1 Summary of distribution of staining intensities representing protein expression 545

within (A) biofilm or (B) non-biofilm groups after 12h treatment with 5% (w/v) HICA, 546

10 mg/L caspofungin or PBS. Sections of subcutaneous tissue surrounding the 547

diffusion chamber were stained using polyclonal antibodies against MMP-8, MMP-9, 548

MPO, NE, TNFα, IL1β, and Del-1. Intensity of the staining was analysed semi-549

quantitatively by two evaluators and graded as no staining, mild, moderate or strong 550

staining. 551

552

FIG 2 Representative images of the tissue sections immunostained with polyclonal 553

antibodies for (A) matrix metalloproteinase (MMP-9), (B) myeloperoxidase (MPO) 554

and (C) developmental endothelial locus-1 (Del-1). The serrated line shows the 555

location of the semipermeable membrane of the chamber. Framed panels on the 556

right are magnifications of the areas marked with black rectangles in the left hand 557

panels. The expression of pro-inflammatory proteases MMP-9 and MPO localised 558

predominantly in the inflammatory cells. The staining intensities for MMP-9 and MPO 559

were lower in HICA treated biofilm group compared to controls (caspofungin and 560

PBS) and histopathology was similar to non-biofilm controls with a thinner and less 561

dense inflammatory cell infiltrate (A,B). Expression of neutrophil extravasation 562

antagonist Del-1 localised in the endothelium and was stronger in the HICA 563

treatment group (C). 564

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