The origin of extracellular DNA in bacterial biofilm infections in vivo · 76 biofilm formation and...

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1 The origin of extracellular DNA in bacterial biofilm infections in vivo

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3 Maria Alhede1, Morten Alhede1, Klaus Qvortrup2, Kasper Nørskov Kragh1, Peter

4 Østrup Jensen1,3, Philip Shook Stewart4 and Thomas Bjarnsholt1, 3*

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6 1Costerton Biofilm Center, Department of Immunology and Microbiology, University

7 of Copenhagen, DK-2200 Copenhagen, Denmark (e-mails: [email protected],

8 [email protected], [email protected], [email protected])

9 2 CFIM/Department of Biomedical Sciences, University of Copenhagen, DK-2100

10 Copenhagen, Denmark (e-mail: [email protected])

11 3Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen,

12 Denmark ([email protected])

13 4Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717-

14 3980, USA ([email protected])

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16 Running title: eDNA is not observed within in vivo biofilms171819 *Corresponding author

20 Thomas Bjarnsholt, Professor, dr. med & Ph.D.

21 University of Copenhagen

22 Faculty of Health Sciences

23 Department of Immunology and Microbiology

24 Costerton Biofilm Center

25 Blegdamsvej 3B,

26 DK-2200 Copenhagen

27 Denmark

28 &

also made available for use under a CC0 license. certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is

The copyright holder for this preprint (which was notthis version posted July 1, 2019. . https://doi.org/10.1101/689067doi: bioRxiv preprint

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29 H:S Rigshospitalet

30 Department for Clinical Microbiology, afsnit 9301,

31 Juliane Maries Vej 22,

32 DK-2100 Copenhagen Ø

33 Denmark

34 @TBjarnsholt

35 Mobile +45 20 65 98 88

36 fax + 45 35 45 64 12

37 [email protected]

38 www.sund.ku.dk

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41424344454647484950

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52 Abstract

53 Extracellular DNA (eDNA) plays an important role in both the aggregation of bacteria

54 and in the interaction of the resulting biofilms with polymorphonuclear leukocytes

55 (PMNs) during an inflammatory response. Here, transmission electron and confocal

56 scanning laser microscopy were used to examine the interaction between biofilms of

57 Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from



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58 chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of

59 PMN DNA with a thymidine analogue, and immunohistochemistry were applied to

60 localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3),

61 neutrophil elastase (NE), and citrullinated H3 (citH3). Host-derived eDNA was

62 observed surrounding bacterial aggregates but not within the biofilms. H3 localized to

63 the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for

64 neutrophil extracellular traps (NETs) was detected only sporadically indicating that

65 most host-derived eDNA in vivo was not a result of NETosis. Together these

66 observations show that, in these in vivo biofilm infections with P. aeruginosa, the

67 majority of eDNA is found external to the biofilm and derives from the host.

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69

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71 Keywords: polymorphonuclear leukocytes/ neutrophil extracellular traps/ NETosis,

72 /Histone/ elastase

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74 Author summary

75 The role of extracellular DNA (eDNA) has been described in vitro to play a major role in 76 biofilm formation and antibiotic tolerance, but never for biofilm infections in vivo.7778 Two important characteristics of human chronic bacterial infections are aggregated 79 bacteria and white blood cells (WBC). Bacteria use eDNA to stabilize biofilms and WBC 80 use eDNA to trap bacteria. Given the importance of eDNA for both bacteria and WBC we 81 show here for the first time that bacterial biofilms do not co-localize with either bacterial 82 or WBC-derived eDNA during chronic infections. Our in vivo findings show eDNA is 83 located outside biofilms as opposed to incorporated within the biofilm as commonly 84 observed in vitro. 8586 We believe that understanding the interplay between biofilms and WBC during chronic 87 infection is essential if we are to elucidate the mechanisms underlying persistent infection 88 and ascertain why WBC fail to eradicate bacteria; this will enable us to develop new 89 treatment strategies.



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909192

93 Introduction

94 Persistent bacterial infections present an increasing challenge to health care

95 practitioners worldwide. Aggregated bacteria, often referred to as biofilms, are tolerant

96 and resistant to both antibiotics and the host immune system [1, 2]. Most studies of

97 bacteria are based on planktonic cultures consisting primarily of single cells grown in

98 vitro where both aggregates and host factors are missing [3]. To understand the

99 underlying mechanism(s) causing persistence of chronic bacterial infections, it is

100 important to investigate the interplay between aggregated bacteria and the host immune

101 system. A common denominator of both bacterial biofilms and cells of the innate host

102 defense is extracellular DNA (eDNA). In vitro studies have shown eDNA, produced by

103 the bacteria themselves, to be important primarily for initial biofilm formation as a

104 major structural component of bacterial biofilms [4] and as a stabilizer that contributes

105 to increased tolerance of biofilms to antibiotics in vitro [5-8]. The common feature of

106 chronic bacterial infections is persistent inflammation dominated by

107 polymorphonuclear leukocytes (PMNs), which border bacterial aggregates but are

108 unable to eradicate them [9]. This type of inflammation is classified as acute

109 inflammation. Successful resolution of acute inflammation involves apoptosis and

110 engulfment of demised PMNs by macrophages [10]. However, during chronic bacterial

111 infections, PMNs are thought to undergo necrosis, during which release of toxic

112 compounds such as NE, oxidants, and eDNA actually increase inflammation [10].

113 Furthermore, components such as NE contribute to the destruction of tissue in chronic

114 wounds and at sites of inflammation in the lungs of cystic fibrosis (CF) patients [11-

115 15]. PMNs actively secrete neutrophil elastase (NE) and histones attached to DNA, via



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116 a process termed NETosis [16], both in vitro and in vivo in acute infections [17, 18].

117 This process is collectively referred to as neutrophil extracellular traps (NETs), which

118 capture and kill bacteria. Since 2004, formation of NETs has been proposed as a

119 function of neutrophils and other immune cells [19] activated in vitro and in vivo in

120 response to various bacterial components [18, 20]. However, a recent review questioned

121 whether NETs play a role in innate immunity and thereby trapping of bacteria [21].

122 Release of eDNA by NETosis has, to the best of our knowledge, never been

123 demonstrated in association with inflammation caused by chronic bacterial infections,

124 despite evidence of close contact between PMNs and bacteria.

125 At present, our knowledge about the interaction between in vivo biofilms and the host

126 immune system during chronic infections is limited, and we do not know the role or

127 localization of eDNA in relation to the biofilms in vivo. Unsuccessful eradication of

128 infectious biofilms despite accumulation of PMNs has not been explained fully. Thus,

129 to obtain more detailed information about the interplay between bacterial aggregates

130 and PMNs during chronic infection, and to increase our understanding of the role of

131 eDNA, we studied these phenomena for the first time, directly in a murine implant

132 model of biofilm infection and directly in human samples of chronic infected CF

133 patients. By using transmission electron microscopy (TEM) we were able to study the

134 interaction between PMNs and biofilm of Pseudomonas aeruginosa formed on a

135 silicone implant inserted in the peritoneal cavity of mice. Furthermore, labeling the

136 DNA of PMNs during their S-phase using the Click-iT technology in vivo and confocal

137 scanning laser microscopy (CSLM), showed PMN-derived DNA did not localize with

138 biofilms. To establish the localization of specific components originating from PMNs

139 we used immunohistochemistry and found that PMN-derived histone H3 (H3) and

140 neutrophil elastase (NE) co-localized with biofilms, but citrullinated H3 (citH3) and



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141 PMN-derived DNA did not. The results obtained using the mouse model could be

142 correlated directly to chronic bacterial infections in human CF lungs.

143

144 Results

145 TEM-based examination of the interaction between biofilms and PMNs in a

146 murine implant model shows damaged PMNs

147 Previously, we used scanning electron microscopy (SEM) [22] to examine the

148 interaction between immune cells and P. aeruginosa biofilms in a murine implant

149 model and found a marked influx of PMNs toward the biofilms. We then used CSLM

150 to identify PMNs according to their segmented lobular nucleus. Here, we used TEM to

151 examine the in-depth additional detailed interaction between bacteria and PMNs, and

152 to visualize the matrix of the biofilm. Implants were inspected at three different time

153 points (6 h, 24 h, and 48 h post-insertion) to illustrate progression of infection and the

154 immune response.

155 At 6 h post-insertion, we noted intact PMNs containing internalized bacteria within

156 vacuoles, which indicates active phagocytosis (Fig 1A). PMNs with internalized

157 bacteria were confined to areas in which bacteria had not yet aggregated. No intact

158 PMNs were observed in areas of high bacterial density (Fig 1B). At 24 h post-insertion,

159 bacterial aggregation resulted in the formation of large biofilms lining the inside of the

160 implants. PMNs frequently adjoined the bacteria. These PMNs were enlarged and had

161 damaged cell membranes (Fig 1C). Intact PMNs at this time point were rare. Matrix

162 material surrounding the bacteria within the biofilm was evident (Fig 1 E-G). At 48 h

163 post-insertion, the biofilm appeared denser and the PMNs were tightly interfaced with

164 the biofilms (Fig 1D). Matrix material was clearly visible (Fig S1).



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165

166 In vitro interaction of P. aeruginosa and PMNs results in lysis of PMNs

167 When we examine the in vitro interaction between PMNs and aggregated P. aeruginosa

168 microscopically, time series reveal that PMNs lyse resulting in release, expansion and

169 dissolution of DNA (Movie 1). The staining of the nuclear content, DNA, with

170 propidium iodide (PI), increases over time as the membrane of the PMNs becomes

171 damaged. At the end the DNA disappears (the red color from PI fades) (Movie 1).

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173 PMN-derived eDNA surrounds in vivo biofilms in a murine implant model

174 To examine eDNA in vivo, we stained silicone implants ex vivo from the murine

175 implant model with SYTO9 (green) which binds to DNA together with the Click-iT®

176 DNA labeling technology kit to determine the origin (i.e., murine or bacterial) of the

177 eDNA in biofilms in vivo. This method utilizes a modified thymidine analogue, 5-

178 ethynyl-2'-deoxyuridine (EdU), which is incorporated into DNA during active DNA

179 synthesis in the S-phase of the eukaryotic cell cycle. Ex vivo, PMN DNA was detected

180 by labeling with a fluorescent Alexa Fluor® dye (pink). Treatment of healthy mice

181 showed the presence of EdU labeled PMNs in the blood 2-5 days after treatment. Since

182 the release of PMNs into the blood increases during an infection [23], we decided to

183 treat the mice two days pre-infection to make sure EdU labeled PMNs would reach the

184 implant and biofilm post-infection. In addition to bacteria aggregated within biofilms

185 (Fig 2 and 3), long strings of eDNA (green) were clearly visible in the PMN

186 accumulations surrounding the biofilms, but not as part of the biofilm itself (Fig 2 and

187 3).



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188 As seen in Fig 2, eDNA strings labeled with pink dye are clearly visible in close

189 proximity to PMNs 24h post-insertion of the implant. However, no pink eDNA was

190 detected in areas occupied by bacterial biofilms (Fig 3 and 4) at 24h or 48h post-

191 insertion, indicating that PMN-derived eDNA is not present internally within these

192 structures.

193 eDNA surrounds in vivo biofilms in lung tissue of chronically infected CF patients

194 Previously, peptide nuclear acid (PNA) fluorescence in situ hybridization (FISH) and

195 4’,6-diamidino-2-phenylindole (DAPI) have been used to identify bacterial aggregates

196 surrounded by PMNs in paraffin-embedded sections of CF lung tissue [9]. The PNA

197 probes bind to bacterial ribosomal 16S RNA [24] and DAPI binds to the A-T regions

198 of double-stranded DNA. Here we used PNA FISH in combination with DAPI to show

199 that eDNA (blue) was localized external to the biofilm in CF lung tissue (Fig 5). The

200 biofilm stained red by a Texas Red-labeled P. aeruginosa-specific PNA probe (Fig 5A).

201 The nuclei of PMNs were clearly stained, as were the strings of DNA outside the

202 biofilm (Fig 5B).

203 Localization of histone H3 (H3), citrullinated H3 (citH3), and NE in a murine

204 implant model

205 To localize components originating from murine PMNs during a bacterial biofilm

206 infection, we stained paraffin-embedded sections of silicone implants isolated 24 h

207 post-insertion in the peritoneal cavity of mice with antibodies specific for citH3 (Fig

208 6), H3 (Fig 7), or NE (Fig 8). All of these components, especially citH3, have also been

209 associated with NETs. Hence, we used citH3 as a marker for NETs [25-28].

210 H3 localized to areas surrounding bacterial biofilms. Only weak staining was observed

211 inside the biofilm; these findings are in agreement with those described above (i.e., no



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212 eDNA originating from PMNs was observed within biofilms). citH3 localized with

213 some PMNs, but the lack of citH3 signifies that the eDNA is not generated by NETosis.

214 In contrast staining with specific antibodies against NE revealed distinct co-localization

215 of biofilms and NE, indicating that NE is incorporated into biofilms (Fig 8). Control

216 images are presented in Fig S4A.

217 Localization of H3, citH3, and NE in lung tissue of chronically infected CF patients

218 Co-localization of NE and H3 with P. aeruginosa biofilms in the murine implant model

219 led us to examine paraffin-embedded lung sections from explanted CF patients with

220 chronic bacterial infections. To verify function and correct binding of the three

221 antibodies when using human PMNs, we stained in vitro phorbol myristate acetate

222 (PMA) generated NETs from human PMNs. We were able to show co-localization of

223 NE and citH3 with NETs, but not with H3 (Fig S2). The lack of H3 co-localization

224 correlates well with the fact that NETosis results in a partial degradation of H3 and

225 posttranslational histone modifications and when using PMA as stimulation a total loss

226 of full length H3 is observed [20, 29]. However, we did find co-localization of H3 with

227 the nucleus of unstimulated PMNs, which indicates a functional antibody (Fig S3). NE

228 also co-localized with the cytoplasma of unstimulated PMNs of paraffin-embedded

229 sections (Fig S3).

230 In CF lungs the distribution of citH3, H3 and NE was similar to that observed in the

231 murine implant model. CitH3 was observed in a few PMNs but did not co-localize with

232 bacterial aggregates (Fig 9A-C). H3 was observed at the periphery of biofilms but not

233 co-localized with the biofilm (Fig 9D-F). NE co-localized with bacteria within the

234 biofilm (Fig 9G-I). Control images are shown in Fig. S4B. To estimate the co-

235 localization of NE, H3 and citH3 with in vivo biofilms we used Manders’ co-

236 localization coefficient as seen in Fig 9J. A value of 1, means that the green pixels



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237 (SYTO9) are perfectly co-localized with the pink pixels (antibody). The co-localization

238 coefficient confirmed what we visually observed, H3 localized outside of biofilms

239 (p<0.0001), citH3 sometimes to outside of biofilms (p<0.0007) and NE localized

240 primarily to biofilms (p<0.25). There was not a significant difference between the

241 outside and biofilm areas for NE. Some of the biofilms seemed to be located in a

242 substance material, presumably alginate as previously shown [9] due to mucoid P.

243 aeruginosa. These biofilms did not co-localize with NE. Instead NE was co-localized

244 with the surrounding PMNs.

245 Discussion

246 Surprisingly, our findings show that, in vivo, eDNA is concentrated external to

247 bacterial aggregates rather than inside as shown previously in vitro by us and others

248 [1, 4]. In fact, the lack of SYTO9 and DAPI staining within the bacterial aggregates in

249 chronic biofilm infections truly questions the significance of the, otherwise in vitro

250 important, role of eDNA in biofilms in vivo as a scaffold of the biofilm. Instead it

251 highlights the important and detrimental feature of host-derived eDNA as a

252 protective shell surrounding the bacteria. This barrier of host eDNA surrounding a

253 biofilm in vivo, which could both limit dissemination of bacteria and shield the

254 biofilm from phagocytosis, may be manifested by necrotic lysis of PMNs rather than

255 through NETosis. Thus, our results are in support of deposition of PMN content in a

256 zone between the PMNs and the biofilm as recently demonstrated in experimental P.

257 aeruginosa ocular biofilm infection where the deposition of NETosis derived material

258 prevented dissemination [30]. In our study, we found only scarce amount of host-

259 derived eDNA and citH3 suggesting a modest contribution by NETosis to the material

260 in the zone between PMNs and biofilm. However, dissemination of the bacteria may



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261 be prevented by the NE localized to the biofilm and therefore we propose other

262 mechanisms than NETosis to contribute to the stalemate and chronicity of biofilm

263 infection as illustrated in Fig. 10.

264 Using TEM we observed biofilm formation and swollen PMNs with damaged

265 membranes, which is in agreement with our former SEM observations [22] showing

266 PMNs failing to eradicate bacterial aggregates. The matrix material seen between the

267 bacteria in the biofilm has not been fully characterized, but in vitro studies show the

268 importance of eDNA for initial formation of young surface biofilms in vitro [4];

269 accordingly, it is believed to be an important structural component [6, 8]. Furthermore,

270 P. aeruginosa has been shown to be attached to eDNA in CF sputum [31] and

271 incorporate external eDNA into the biofilm matrix in vitro [5]. PMNs were enlarged

272 and had damaged cell membranes, presumably due to exposure to the bacterial

273 virulence factor, rhamnolipid, which is detrimental to PMNs [32, 33]. It has previously

274 been shown that P. aeruginosa produce rhamnolipids which cause PMNs to swell and

275 burst, thereby releasing their cytoplasmic and nuclear content into the surrounding area

276 [34]. These cellular components including eDNA from necrotic PMNs are believed to

277 serve as a biological matrix for biofilm formation and a protective shield within the

278 biofilm increasing tolerance to treatment to tobramycin in vitro [5, 31]. This was

279 confirmed in a mouse model where a rhlA mutant (unable to excrete rhamnolipid) did

280 not cause any PMN lysis and no release of cytoplasmic and nuclear content, thus

281 consistent with a mechanism of lysis by the biosurfactant rather than a

282 programmed NET pathway for eDNA release [32].

283 The lack of strings in in vivo biofilms indicate that unlike in vitro biofilms, eDNA is

284 not a direct part of the biofilm. Visually eDNA from P. aeruginosa has been shown to

285 form a net both in a stationary grown biofilm and in the flow cell biofilm [1, 8].



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286 Furthermore, eDNA from P. aeruginosa is believed to be double-stranded DNA [8] and

287 since DAPI bind to double-stranded DNA the lack of DAPI staining signifies the lack

288 of eDNA in the in vivo biofilm. Also, the measurement of double-stranded DNA has

289 been shown to decrease when P. aeruginosa change from the planktonic state to the

290 aggregating state [35]. However, the eDNA of a plate grown colony of P. aeruginosa

291 have been shown to be 5 m long [36] which mean that visually the eDNA fragment

292 would be almost as small as a single P. aeruginosa bacterium. Visually this does not

293 correlate to our in vivo and others in vitro findings. However, we might have to rethink

294 the need for a structural matrix depending on the environment of the biofilm outside

295 the laboratory. Maybe when the biofilm is exposed to high forces such as fluid it needs

296 to establish a solid structure, but in the CF lung another type of structure is established

297 within the mucus to withstand the immune cells. Accordingly, this indicates that neither

298 bacterial nor PMN-derived eDNA is incorporated into biofilms in vivo, but eDNA from

299 necrotic PMNs is more likely accumulating outside the biofilms.

300 Since we only found host-derived eDNA outside infectious biofilms in vivo, we

301 speculated whether in vivo biofilm matrix structures comprised other components

302 originating from PMNs (e.g., H3, citH3, or NE). PMNs undergoing apoptosis, necrotic

303 cell death, or NETosis release different histones (i.e., H3 during necrotic cell death and

304 citH3 during NETosis), either into the surrounding area or attached to NETs [17].

305 Release of these extracellular histones promotes secretion of pro-inflammatory

306 cytokines and recruitment of immune cells to sites of infection [37]. Histones bind to

307 lipopolysaccharide (LPS) derived from P. aeruginosa, which neutralizes the positive

308 charge of the histone molecule [38]; with the outcome of antimicrobial activity [39].

309 The fact that histones are antimicrobial [17, 40], might not be the case in this study.

310 Since H3 is rich in arginine it may rather play a role in the arginine pathway in P.



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311 aeruginosa, which breaks down arginine to ornithine via the arginine deiminase

312 pathway to sustain growth under anoxic conditions. Ornithine is a carbon source used

313 to maintain growth during periods in which nitrate is either limited or lacking [41-43].

314 Arginine has also been shown to contribute to biofilm formation [44].

315 Here, we showed that H3 is mostly present along the periphery of biofilm aggregates,

316 but not within the biofilms. This could indicate that H3 is still bound to eDNA. By

317 contrast, citH3 did not localize with biofilms in either the mouse model or CF lung

318 samples. CitH3 is a post-translational modification of H3. Conversion of arginine to

319 citrulline is accomplished by peptidylarginine deiminase 4 (PAD4) and is a prerequisite

320 for in vitro generated NETosis since the modification of arginine residues to citrulline

321 changes the charge of the histones, leading to massive chromatin de-condensation,

322 NETs [26, 27, 45]. NETs are highly de-condensed chromatin structures that comprise

323 mainly DNA, although histones and antimicrobial granular substances such as NE are

324 also present [17]. Histone citrullination in vitro depends on stimulation of PMNs [46,

325 47]; indeed, an increase in histone citrullination is associated with chromatin de-

326 condensation during NETs formation and LPS-induced early responses to

327 inflammatory stimuli of PMNs [25, 26]. However, one widely used stimulus, PMA, has

328 resulted in conflicting results in the literature and has been shown to induce NETs with

329 and without co-localized citH3 and H3 [17, 47, 48]. In addition citrullination of H3 is

330 strongly reduced when PMNs are stimulated during anoxic conditions [49], which is

331 present in the endobronchial secretion in infected CF lungs [50, 51].

332 The lack of host-derived eDNA and citH3 associated with bacterial aggregates in the

333 present study demonstrates that NETosis probably does not occur in chronic biofilm

334 infections. Release of DNA under these conditions is not an active process as in

335 NETosis; rather, it is more likely due to lysis of PMNs [34] which correlates to the



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336 presence of H3 and absence of citH3. In addition, our findings that NETosis seemingly

337 plays no role in chronic P. aeruginosa infections correlates to the depletion of

338 molecular oxygen at the site of chronic biofilm infections [52], thereby preventing

339 NADPH oxidase from generating the reactive oxygen species (ROS) needed to release

340 NETs [53]. Thus, PAD4-dependent NET formation might not be responsible for DNA

341 release in CF airways and we speculate whether citH3 may serve as a biomarker for

342 discriminating chronic from acute infections. However, NET generation independent

343 of ROS generated by NADPH oxidase has been shown in vivo [54] therefore further

344 work is needed to confirm this. Nevertheless, eDNA in sputum samples from CF

345 patients have been shown to originate from PMNs [55] with NETs as the major

346 component [56]. However, since NETs are antimicrobial this should benefit the CF

347 patients since this should lead to eradication of the present bacteria. On the contrary

348 eDNA has been shown to increase the viscosity of the mucus and bind antibiotics such

349 as tobramycin [57, 58]. Furthermore, by treating the CF patients with DNase an

350 improved lung function in CF patients and an increased effect of antibiotics have been

351 reported [59, 60].

352 We did observe NE within biofilms, although this may have been derived from a

353 necrotic cell death of PMNs external to the biofilm, followed by passive diffusion into

354 the aggregate structure. NE is stored in primary (azurophilic) granules and released

355 upon degranulation, phagocytosis, and cell death. NE cleaves a wide variety of proteins,

356 including bacterial virulence factors [61]. The purpose of NE is to degrade

357 phagocytosed proteins; however, in CF patients NE damages the airways [62].

358 Furthermore, NE is an important component of NETs [17]. However, since NE was

359 found within the biofilms and eDNA was not supports the lack of NETs, since in these

360 structures NE is bound to NETs. Furthermore, unlike in acute infections caused by



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361 Gram-negative bacteria P. aeruginosa is not killed by NE following aggregation and

362 biofilm formation [63]. The absence of NE-mediated killing is likely attributed to

363 production of the protease inhibitor ecotin by bacteria [64]. In addition, NET formation

364 induced by P. aeruginosa depends strongly on flagella motility [65], which may further

365 explain the absence of NETs in CF lungs in which the flagella of P. aeruginosa are

366 down-regulated [66] and in which flagellin, which is the major component of flagella,

367 may be cleaved by NE [67]. Since a decrease and/or absence of flagella motility is

368 associated with formation of bacterial biofilms [68], a lack of motile flagella may lead

369 to a lack of detectable NETs.

370 In conclusion, this study is the first to examine the direct distribution of host and

371 bacterial eDNA during chronic bacterial infections in vivo. Unlike after stimulation in

372 vitro, stimulation of PMNs by bacterial aggregates during chronic infections in vivo

373 does not seem to trigger active release of NETs; however, necrotic PMNs do release

374 eDNA, histone H3, and antibacterial enzymes such as NE.

375 We did not find evidence of eDNA being part of in vivo biofilms, instead the PMN-

376 derived layer of eDNA, which constitutes a secondary matrix, may provide a passive

377 physical shield for the biofilm against cationic antibiotics such as tobramycin and

378 additional phagocytes. A similar spatial arrangement has recently been described in a

379 murine model of P. aeruginosa infection of the cornea in which the bacterial biofilm

380 and neutrophil layers are separated by a “dead zone” rich in eDNA and NE [69].

381 Thus, we believe that eDNA released by PMNs due to a necrotic cell death contribute

382 to the protection afforded to bacteria by biofilms (Fig. 10). These novel findings shed

383 new light on the origin and role of eDNA in in vivo biofilms and unexpectedly question

384 the role of extracellular host DNA during inflammation associated with chronic

385 bacterial infections and of NET activation.



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386

387 Materials and Methods

388 Murine implant model

389 All experiments were performed using a wild-type (WT) P. aeruginosa strain obtained

390 from Professor Barbara Iglewski (University of Rochester Medical Center, NY, USA).

391 The strain is QS proficient, except for the reduced production of C4-HSL previously

392 noted for this P. aeruginosa variant [70]. Bacteria obtained from freezer stocks were

393 plated onto blue agar plates (State Serum Institute, Denmark) and incubated overnight

394 at 37°C. Blue agar plates are used to select Gram-negative bacilli [71]. One colony was

395 used to inoculate overnight cultures grown in Luria-Bertani (LB) medium at 37°C with

396 shaking at 180 rpm. Female BALB/c mice (6 weeks-of-age) were purchased from

397 Taconic M&B A/S (Ry, Denmark) and maintained on standard mouse chow and water

398 ad libitum for 2 weeks before challenge. The murine implant model was generated as

399 described previously [22, 72], with some modifications. Briefly, the silicon tubes (Ole

400 Dich Instrumentmakers Aps. Silicone Pumpeslange 60 Shore A (ID: 4.0 mm, OD: 6.0

401 mm, wall: 1.0 mm) were cut to a length of 4 mm, and the bacterial pellet from a

402 centrifuged overnight culture was resuspended in 0.9% NaCl (OD600nm = 0.1). Mice

403 were anesthetized by subcutaneous (s.c.) injection of hypnorm/midazolam (Roche)

404 [hypnorm (0.315 mg ml-1 fentanyl citrate; 10 mg ml-1 fluanisone); midazolam (5 mg

405 ml-1); and sterile water; 1:1:2 v/v]. The pre-coated silicone implants were inserted in

406 the peritoneal cavity of the mice. The mice were treated with bupivacaine and

407 Temgesic® as post-operative pain relief. Experiments were terminated with

408 euthanization of the mice with intraperitoneal injection of pentobarbital (DAK; 10.0 ml

409 kg-1 body weight).



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410 Preparation of samples for TEM

411 The silicone implants were fixed in 2% glutaraldehyde in 0.05 M sodium phosphate

412 buffer pH 7.4. Growing biofilms on a silicone tube presents a challenge with respect to

413 sample preparation prior to the different microscopy techniques. First, a traditional

414 preparation method was used for TEM to get an indication of the interaction between

415 PMNs and the biofilm. However, some of the steps involved in the embedding process

416 were shortened. Samples were observed with a CM 100 TEM (Philips, the Nederlands)

417 operated at 80kV. Imaged were recorded with a side-mounted Olympus Veleta camera

418 (resolution, 2048 × 2048 pixels (2K × 2K)) and the iTEM (Olympus, Germany)

419 software package.

420 In vitro experiments with PMNs and P. aeruginosa

421 Wild-type P. aeruginosa (strain PAO1) was obtained from the Pseudomonas Genetic

422 Stock Center (www.pseudomonas.med.ecu.edu; strain PAO0001). Bacteria were

423 tagged with a stable green fluorescent protein (GFP) constitutively expressed by

424 plasmid pMRP9 [73]. PAO1 cultures were grown for 24 h at 37°C in 12 well microtiter

425 plates containing LB medium. Human blood was collected from healthy volunteers

426 with the approval of the Danish Scientific Ethical Board (H-3-2011-117). PMNs were

427 isolated as described [74]. Isolated PMNs were resuspended in krebs ringer buffer

428 containing 10mM glucose at 37°C (final density, 2.5 × 107 PMNs/ml). Propidium

429 iodide (2.5 ug/ml) was used as an indicator of cell death. PAO1 (350 l) and PMNs (50

430 l) were mixed in a 12 well microtiter plate and observed under a confocal microscope

431 for 35 min.



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432 Click-iT™

433 Mice were treated with EdU (Click-iT™ EdU Alexa Fluor™ 647 Imaging Kit)

434 (C10640, ThermoFisher) for 2 days pre-infection and until termination of the

435 experiment. Each mouse was injected i.p. twice a day with 25 mg/g EdU dissolved in

436 0.9% NaCl. Control mice received 0.9% NaCl without EdU. Ex vivo implants were

437 fluorescently labeled using the Click-iT kit, according to the manufacturer’s

438 instructions, and stained with SYTO9 (S34934, ThermoFisher) prior to observation

439 under a microscope.

440 In vitro generated NETs

441 In vitro NETs were generated according to Vong et. al. [75] and Brinkmann et. al. [76]

442 with modifications. PMNs were isolated according to Hu et. al. [77] using

443 polymorphprep with modifications. Isolated PMNs were suspended in RPMI 1640 with

444 glutamine and were seated in an ibidi slide coated with poly-L-lysine (80604, ibidi) for

445 30 min before stimulation with PMA for 3h. The PMNs were incubated at 37 degrees

446 and 5% CO2. PMNs was washed with PBS and fixed in 4% PFA pH 7.2 for 15 min,

447 washed in 0.025% Triton X-100 twice before proceeding with antibody staining as

448 described for paraffin sections. Before imaging the channels were filled with Ibidi

449 mounting medium (50001, ibidi).

450 Ex vivo paraffin samples

451 Samples from the mouse model, the lungs of CF patients, and unactivated human PMNs

452 were fixed in 4% formalin and paraffin-embedded according to standard protocols.

453 Sections (4 m thick) were cut using a standard microtome, fixed on glass slides, and

454 stored at 4°C until required.

455



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456 Lung tissue from chronically infected CF patients

457 Explanted lungs tissue from 3 contemporary intensively treated P. aeruginosa infected

458 CF patients were examined [9].

459 PNA FISH

460 PNA FISH staining was performed as previously described [9, 78]. Texas Red-labeled

461 P. aeruginosa-specific PNA probe both in hybridization solution (AdvanDx, Inc.,

462 Woburn, MA), was added to each section and hybridized in a PNA FISH workstation

463 covered by a lid at 55°C for 90 min. The slides were washed for 30 min at 55°C in wash

464 solution (AdvanDx) and counterstained with DAPI (D1306, ThermoFisher).

465 Antibody staining of paraffin-embedded tissue sections

466 Sections were deparaffinized in 99.9% xylene (twice for 5 min each time) and

467 subsequently hydrolyzed in 99.9% ethanol (twice for 5 min each time), followed by

468 96.5% ethanol (twice for 3 min each time). Finally, sections were rinsed in water (twice

469 for 5 min each time). Heat-induced antigen retrieval was performed in citrate buffer,

470 pH 6.0 (Sigma C9999) for 15 min in a domestic microwave oven. For unstimulated

471 human PMNs a permabilization step was included using 0.1% Triton X-100 for 20 min.

472 Samples were washed twice (5 min each time) in 0.025% triton X-100, drained, and

473 blocked for 2 h at room temp in 10% goat serum in 1% BSA/PBS. Samples were

474 drained and incubated for 16–18 h at 4°C with a primary antibody (Abcam Cat#

475 ab1791, RRID:AB_302613, Abcam Cat# ab5103, RRID:AB_304752, or Abcam Cat#

476 ab21595, RRID:AB_446409), diluted 1:200 in 2% goat serum/1% BSA in PBS).

477 Negative controls were isotype IgG (Abcam Cat# ab27478, RRID:AB_2616600) or

478 absence of primary antibody. Samples were drained and washed in 0.025% Triton X-

479 100 (twice for 5 min each) with gentle agitation. Samples were then incubated (1 h at



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480 room temp in the dark) with a secondary antibody (Abcam Cat# ab150083,

481 RRID:AB_2714032) diluted 1:1000 in 1% BSA/PBS. Finally, samples were washed 3

482 times in PBS (5 min each time) and counterstained with SYTO9 (S34934,

483 ThermoFisher). ProLong™ gold antifade mountant (Thermofisher, P36934) was

484 applied before adding the coverslip.

485

486 Co-localization coefficient

487 Co-localization refers to the geometric co-distribution of two fluorescent labels or color

488 channels. Co-localization was acquired in Zen (version 2.1 SP3 FP2 (black)) as

489 described by Zeiss “Acquiring and analyzing data for co-localization experiments in

490 AIM or Zen software”. The Manders’ co-localization coefficient [79] for SYTO9

491 (green channel ) was used to show the co-localization of the antibody (pink channel)

492 using 2D images. Perfect co-localization will give the value 1. The crosshair was set to

493 1003 for the green channel and 2500 for the pink channel based on single stained

494 paraffin sections according to the manual by Zeiss. Sections from 3 different patients

495 were used. Biofilm and PMN areas were selected by region of interest (ROIs) in each

496 paraffin section. All biofilm areas in one image were included. GraphPad Prism was

497 used for statistical analysis using unpaired t-test. P<0.05 was considered significant.

498

499 Confocal scanning laser microscopy

500 All observations and image acquisition were performed using a confocal scanning laser

501 microscope (LSM510, LSM 710, or LSM 880; Carl Zeiss GmbH, Germany). Images

502 were obtained using 40× 63× or 100× oil objective lenses. Images were scanned at 405

503 nm (blue), 488 nm (green), and 633 nm (far red). Images of the biofilms were generated



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504 using Imaris software including the magnifications (version 8.4, Bitplane AG). Images

505 were processed for display using Photoshop software (Adobe).

506507 Ethics Statement

508 All animal studies were carried out in accordance with the European convention and

509 Directive for the Protection of Vertebrate Animals used for Experimental and Other

510 Scientific Purposes, and with Danish law on animal experimentation. All experiments

511 were authorized and approved by the National Animal Ethics Committee, Denmark

512 (The Animal Experiments Inspectorate, dyreforsoegstilsynet.dk; permit numbers:

513 2012−15−2934−00677 and 2014−15−0201−00259).

514

515 Human blood was collected from healthy volunteers with the approval of the Danish

516 Scientific Ethical Board (H-3-2011-117). All donors provided written informed

517 consent.

518

519 Infected tissue was collected following approval by the Danish Scientific Ethical

520 Board (KF465 01278432). The samples used were collected with written informed

521 consent for a previous study [9], and only from adults. The samples were anonymized

522 for this study.

523

524 Acknowledgments

525 We acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and

526 Medical Sciences, University of Copenhagen, Zhila Nikrozi for preparing samples for

527 TEM, Heidi Marie Paulsen for preparing paraffin sections, and Steen Seier Poulsen for

528 helpful discussions regarding antibody staining procedures.



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529 M.A., M.A., and T.B. were funded by the Lundbeck Foundation. M.A. was also

530 founded by Hørslev-Fonden and Torben and Alice Frimodt Fonden.

531

532 Author contributions

533 M.A. conducted the mouse experiments and antibody staining experiments, and wrote

534 and edited the manuscript.

535 M.A. undertook microscopic analysis of Click-It and edited the manuscript.

536 K.Q. assisted with TEM preparation and image recording, and edited the manuscript.

537 P.Ø.J. performed the in vitro PMN experiments, contributed to data interpretation and

538 edited the manuscript.

539 K.N.K helped with the co-localization, helped with figure 10 and edited the

540 manuscript

541 P.S. contributed to data interpretation and edited the manuscript

542 T.B. performed the in vitro PMN experiments and PNA FISH, contributed to data

543 interpretation and edited the manuscript.

544

545 Conflicts of interest

546 The funders had no role in study design, data collection and analysis, decision to

547 publish, or preparation of the manuscript.

548

549 References

550



https://doi.org/10.1101/689067

23

551 1. Alhede M, Kragh KN, Qvortrup K, Allesen-Holm M, van Gennip M,

552 Christensen LD, et al. Phenotypes of non-attached Pseudomonas aeruginosa

553 aggregates resemble surface attached biofilm. PLoS One. 2011;6(11):e27943. Epub

554 2011/12/02. doi: 10.1371/journal.pone.0027943. PubMed PMID: 22132176; PubMed

555 Central PMCID: PMC3221681.

556 2. Bjarnsholt T, Alhede M, Alhede M, Eickhardt-Sørensen SR, Moser C, Kuhl

557 M, et al. The in vivo biofilm. Trends Microbiol. 2013. Epub 2013/07/06. doi:

558 10.1016/j.tim.2013.06.002. PubMed PMID: 23827084.

559 3. Moser C, Pedersen HT, Lerche CJ, Kolpen M, Line L, Thomsen K, et al.

560 Biofilms and host response - helpful or harmful. APMIS. 2017;125(4):320-38. doi:

561 10.1111/apm.12674. PubMed PMID: 28407429.

562 4. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA

563 required for bacterial biofilm formation. Science. 2002;295(5559):1487. Epub

564 2002/02/23. doi: 10.1126/science.295.5559.1487. PubMed PMID: 11859186.

565 5. Chiang WC, Nilsson M, Jensen PØ, Høiby N, Nielsen TE, Givskov M, et al.

566 Extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa

567 biofilms. Antimicrob Agents Chemother. 2013. Epub 2013/03/13. doi:

568 10.1128/AAC.00001-13. PubMed PMID: 23478967.

569 6. Izano EA, Amarante MA, Kher WB, Kaplan JB. Differential roles of poly-N-

570 acetylglucosamine surface polysaccharide and extracellular DNA in Staphylococcus

571 aureus and Staphylococcus epidermidis biofilms. Appl Environ Microbiol.

572 2008;74(2):470-6. doi: 10.1128/AEM.02073-07. PubMed PMID: 18039822; PubMed

573 Central PMCID: PMCPMC2223269.

574 7. Das T, Sehar S, Manefield M. The roles of extracellular DNA in the structural

575 integrity of extracellular polymeric substance and bacterial biofilm development.



https://doi.org/10.1101/689067

24

576 Environ Microbiol Rep. 2013;5(6):778-86. doi: 10.1111/1758-2229.12085. PubMed

577 PMID: 24249286.

578 8. Allesen-Holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S, et

579 al. A characterization of DNA release in Pseudomonas aeruginosa cultures and

580 biofilms. Mol microbiol. 2006;59(4):1114-28. Epub 2006/01/25. doi: 10.1111/j.1365-

581 2958.2005.05008.x. PubMed PMID: 16430688.

582 9. Bjarnsholt T, Jensen PØ, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB,

583 et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis

584 patients. Pediatr Pulmonol. 2009;44(6):547-58.

585 10. Cox G, Crossley J, Xing Z. Macrophage engulfment of apoptotic neutrophils

586 contributes to the resolution of acute pulmonary inflammation in vivo. Am J Respir

587 Cell Mol Biol. 1995;12(2):232-7. Epub 1995/02/01. PubMed PMID: 7865221.

588 11. Ohlsson K, Olsson I. The extracellular release of granulocyte collagenase and

589 elastase during phagocytosis and inflammatory processes. Scand J Haematol.

590 1977;19(2):145-52. PubMed PMID: 197589.

591 12. Voynow JA, Fischer BM, Zheng S. Proteases and cystic fibrosis. The

592 international journal of biochemistry & cell biology. 2008;40(6-7):1238-45. doi:

593 10.1016/j.biocel.2008.03.003. PubMed PMID: 18395488; PubMed Central PMCID:

594 PMCPMC2431113.

595 13. Yager DR, Nwomeh BC. The proteolytic environment of chronic wounds.

596 Wound Repair Regen. 1999;7(6):433-41. PubMed PMID: 10633002.

597 14. Suleman L. Extracellular Bacterial Proteases in Chronic Wounds: A Potential

598 Therapeutic Target? Adv Wound Care (New Rochelle). 2016;5(10):455-63. doi:

599 10.1089/wound.2015.0673. PubMed PMID: 27785379; PubMed Central PMCID:

600 PMCPMC5067851.



https://doi.org/10.1101/689067

25

601 15. McCarty SM, Cochrane CA, Clegg PD, Percival SL. The role of endogenous

602 and exogenous enzymes in chronic wounds: a focus on the implications of aberrant

603 levels of both host and bacterial proteases in wound healing. Wound Repair Regen.

604 2012;20(2):125-36. doi: 10.1111/j.1524-475X.2012.00763.x. PubMed PMID:

605 22380687.

606 16. Steinberg BE, Grinstein S. Unconventional roles of the NADPH oxidase:

607 signaling, ion homeostasis, and cell death. Science's STKE : signal transduction

608 knowledge environment. 2007;2007(379):pe11. Epub 2007/03/30. doi:

609 10.1126/stke.3792007pe11. PubMed PMID: 17392241.

610 17. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS,

611 et al. Neutrophil extracellular traps kill bacteria. Science. 2004;303(5663):1532-5.

612 Epub 2004/03/06. doi: 10.1126/science.1092385

613 303/5663/1532 [pii]. PubMed PMID: 15001782.

614 18. Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD, et al.

615 Infection-induced NETosis is a dynamic process involving neutrophil multitasking in

616 vivo. Nat Med. 2012;18(9):1386-93. Epub 2012/08/28. doi: 10.1038/nm.2847.

617 PubMed PMID: 22922410.

618 19. Goldmann O, Medina E. The expanding world of extracellular traps: not only

619 neutrophils but much more. Frontiers in immunology. 2012;3:420. doi:

620 10.3389/fimmu.2012.00420. PubMed PMID: 23335924; PubMed Central PMCID:

621 PMCPMC3542634.

622 20. Kenny EF, Herzig A, Kruger R, Muth A, Mondal S, Thompson PR, et al.

623 Diverse stimuli engage different neutrophil extracellular trap pathways. Elife. 2017;6.

624 doi: 10.7554/eLife.24437. PubMed PMID: 28574339; PubMed Central PMCID:

625 PMCPMC5496738.



https://doi.org/10.1101/689067

26

626 21. Sørensen OE, Borregaard N. Neutrophil extracellular traps - the dark side of

627 neutrophils. J Clin Invest. 2016;126(5):1612-20. doi: 10.1172/JCI84538. PubMed

628 PMID: 27135878; PubMed Central PMCID: PMCPMC4855925.

629 22. van Gennip M, Christensen LD, Alhede M, Qvortrup K, Jensen PØ, Høiby N,

630 et al. Interactions between Polymorphonuclear Leukocytes and Pseudomonas

631 aeruginosa Biofilms on Silicone Implants In Vivo. Infect Immun. 2012;80(8):2601-7.

632 Epub 2012/05/16. doi: 10.1128/IAI.06215-11. PubMed PMID: 22585963.

633 23. Hansen NE, Karle H, Valerius NH. Neutrophil kinetics in acute bacterial

634 infection. A clinical study. Acta Med Scand. 1978;204(5):407-12. Epub 1978/01/01.

635 PubMed PMID: 717062.

636 24. Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J. PNA for rapid

637 microbiology. J Microbiol Methods. 2002;48(1):1-17. PubMed PMID: 11733079.

638 25. Neeli I, Khan SN, Radic M. Histone deimination as a response to

639 inflammatory stimuli in neutrophils. J Immunol. 2008;180(3):1895-902. PubMed

640 PMID: 18209087.

641 26. Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, et al. Histone

642 hypercitrullination mediates chromatin decondensation and neutrophil extracellular

643 trap formation. J Cell Biol. 2009;184(2):205-13. doi: 10.1083/jcb.200806072.

644 PubMed PMID: 19153223; PubMed Central PMCID: PMCPMC2654299.

645 27. Wong SL, Demers M, Martinod K, Gallant M, Wang Y, Goldfine AB, et al.

646 Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat

647 Med. 2015;21(7):815-9. doi: 10.1038/nm.3887. PubMed PMID: 26076037; PubMed


649 28. Barliya T, Dardik R, Nisgav Y, Dachbash M, Gaton D, Kenet G, et al.

650 Possible involvement of NETosis in inflammatory processes in the eye: Evidence



https://doi.org/10.1101/689067

27

651 from a small cohort of patients. Mol Vis. 2017;23:922-32. PubMed PMID: 29296072;

652 PubMed Central PMCID: PMCPMC5741378.

653 29. Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. Neutrophil

654 elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.

655 J Cell Biol. 2010;191(3):677-91. doi: 10.1083/jcb.201006052. PubMed PMID:

656 20974816; PubMed Central PMCID: PMCPMC3003309.

657 30. Thanabalasuriar A, Scott BNV, Peiseler M, Willson ME, Zeng Z, Warrener P,

658 et al. Neutrophil Extracellular Traps Confine Pseudomonas aeruginosa Ocular

659 Biofilms and Restrict Brain Invasion. Cell host & microbe. 2019;25(4):526-36 e4.

660 Epub 2019/04/02. doi: 10.1016/j.chom.2019.02.007. PubMed PMID: 30930127.

661 31. Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, Saavedra MT, et

662 al. Enhanced Pseudomonas aeruginosa biofilm development mediated by human

663 neutrophils. Infect Immun. 2005;73(6):3693-701. Epub 2005/05/24. doi: 73/6/3693

664 [pii]

665 10.1128/IAI.73.6.3693-3701.2005. PubMed PMID: 15908399; PubMed Central

666 PMCID: PMC1111839.

667 32. van Gennip M, Christensen LD, Alhede M, Phipps R, Jensen PØ,

668 Christophersen L, et al. Inactivation of the rhlA gene in Pseudomonas aeruginosa

669 prevents rhamnolipid production, disabling the protection against polymorphonuclear

670 leukocytes. APMIS. 2009;117(7):537-46. Epub 2009/07/15. doi: APM2466 [pii]

671 10.1111/j.1600-0463.2009.02466.x. PubMed PMID: 19594494.

672 33. Alhede M, Bjarnsholt T, Jensen PØ, Phipps RK, Moser C, Christophersen L,

673 et al. Pseudomonas aeruginosa recognizes and responds aggressively to the presence

674 of polymorphonuclear leukocytes. Microbiology. 2009;155(Pt 11):3500-8. Epub

675 2009/08/01. doi: mic.0.031443-0 [pii]



https://doi.org/10.1101/689067

28

676 10.1099/mic.0.031443-0. PubMed PMID: 19643762.

677 34. Jensen PØ, Bjarnsholt T, Phipps R, Rasmussen TB, Calum H, Christoffersen

678 L, et al. Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-

679 sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa.

680 Microbiology. 2007;153(Pt 5):1329-38. PubMed PMID: 17464047.

681 35. Das T, Sehar S, Koop L, Wong YK, Ahmed S, Siddiqui KS, et al. Influence of

682 calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.

683 PLoS One. 2014;9(3):e91935. Epub 2014/03/22. doi: 10.1371/journal.pone.0091935.


685 36. Chiba A, Sugimoto S, Sato F, Hori S, Mizunoe Y. A refined technique for

686 extraction of extracellular matrices from bacterial biofilms and its applicability.

687 Microb Biotechnol. 2015;8(3):392-403. Epub 2014/08/27. doi: 10.1111/1751-

688 7915.12155. PubMed PMID: 25154775; PubMed Central PMCID:

689 PMCPMC4408173.

690 37. Westman J, Papareddy P, Dahlgren MW, Chakrakodi B, Norrby-Teglund A,

691 Smeds E, et al. Extracellular Histones Induce Chemokine Production in Whole Blood

692 Ex Vivo and Leukocyte Recruitment In Vivo. PLoS pathogens.

693 2015;11(12):e1005319. doi: 10.1371/journal.ppat.1005319. PubMed PMID:

694 26646682; PubMed Central PMCID: PMCPMC4672907.

695 38. Rose-Martel M, Hincke MT. Antimicrobial histones from chicken

696 erythrocytes bind bacterial cell wall lipopolysaccharides and lipoteichoic acids. Int J

697 Antimicrob Agents. 2014;44(5):470-2. doi: 10.1016/j.ijantimicag.2014.07.008.

698 PubMed PMID: 25216546.

699 39. Rose-Martel M, Kulshreshtha G, Ahferom Berhane N, Jodoin J, Hincke MT.

700 Histones from Avian Erythrocytes Exhibit Antibiofilm activity against methicillin-



https://doi.org/10.1101/689067

29

701 sensitive and methicillin-resistant Staphylococcus aureus. Sci Rep. 2017;7:45980. doi:

702 10.1038/srep45980. PubMed PMID: 28378802; PubMed Central PMCID:

703 PMCPMC5380990.

704 40. Hirsch JG. Bactericidal action of histone. The Journal of experimental

705 medicine. 1958;108(6):925-44. PubMed PMID: 13598820; PubMed Central PMCID:

706 PMCPMC2136925.

707 41. Stalon V, Vander Wauven C, Momin P, Legrain C. Catabolism of arginine,

708 citrulline and ornithine by Pseudomonas and related bacteria. J Gen Microbiol.

709 1987;133(9):2487-95. doi: 10.1099/00221287-133-9-2487. PubMed PMID: 3129535.

710 42. Vander Wauven C, Pierard A, Kley-Raymann M, Haas D. Pseudomonas

711 aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene

712 cluster encoding the arginine deiminase pathway. J Bacteriol. 1984;160(3):928-34.


714 43. Eschbach M, Schreiber K, Trunk K, Buer J, Jahn D, Schobert M. Long-term

715 anaerobic survival of the opportunistic pathogen Pseudomonas aeruginosa via

716 pyruvate fermentation. J Bacteriol. 2004;186(14):4596-604. doi:

717 10.1128/JB.186.14.4596-4604.2004. PubMed PMID: 15231792; PubMed Central

718 PMCID: PMCPMC438635.

719 44. Bernier SP, Ha DG, Khan W, Merritt JH, O'Toole GA. Modulation of

720 Pseudomonas aeruginosa surface-associated group behaviors by individual amino

721 acids through c-di-GMP signaling. Res Microbiol. 2011;162(7):680-8. doi:

722 10.1016/j.resmic.2011.04.014. PubMed PMID: 21554951; PubMed Central PMCID:

723 PMCPMC3716369.

724 45. Lewis HD, Liddle J, Coote JE, Atkinson SJ, Barker MD, Bax BD, et al.

725 Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation.



https://doi.org/10.1101/689067

30

726 Nature chemical biology. 2015;11(3):189-91. Epub 2015/01/27. doi:

727 10.1038/nchembio.1735. PubMed PMID: 25622091; PubMed Central PMCID:

728 PMCPMC4397581.

729 46. Neeli I, Radic M. Opposition between PKC isoforms regulates histone

730 deimination and neutrophil extracellular chromatin release. Frontiers in immunology.

731 2013;4:38. doi: 10.3389/fimmu.2013.00038. PubMed PMID: 23430963; PubMed


733 47. Konig MF, Andrade F. A Critical Reappraisal of Neutrophil Extracellular

734 Traps and NETosis Mimics Based on Differential Requirements for Protein

735 Citrullination. Frontiers in immunology. 2016;7:461. doi:

736 10.3389/fimmu.2016.00461. PubMed PMID: 27867381; PubMed Central PMCID:

737 PMCPMC5095114.

738 48. Neeli I, Radic M. Current Challenges and Limitations in Antibody-Based

739 Detection of Citrullinated Histones. Frontiers in immunology. 2016;7:528. Epub

740 2016/12/10. doi: 10.3389/fimmu.2016.00528. PubMed PMID: 27933065; PubMed


742 49. Lopes JP, Stylianou M, Backman E, Holmberg S, Jass J, Claesson R, et al.

743 Evasion of Immune Surveillance in Low Oxygen Environments Enhances Candida

744 albicans Virulence. MBio. 2018;9(6). Epub 2018/11/08. doi: 10.1128/mBio.02120-18.


746 50. Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al.

747 Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of

748 cystic fibrosis patients. The Journal of clinical investigation. 2002;109(3):317-25.

749 PubMed PMID: 11827991.



https://doi.org/10.1101/689067

31

750 51. Kolpen M, Kuhl M, Bjarnsholt T, Moser C, Hansen CR, Liengaard L, et al.

751 Nitrous oxide production in sputum from cystic fibrosis patients with chronic

752 Pseudomonas aeruginosa lung infection. PLoS One. 2014;9(1):e84353. Epub

753 2014/01/28. doi: 10.1371/journal.pone.0084353. PubMed PMID: 24465406; PubMed


755 52. Kolpen M, Hansen CR, Bjarnsholt T, Moser C, Christensen LD, van Gennip

756 M, et al. Polymorphonuclear leucocytes consume oxygen in sputum from chronic

757 Pseudomonas aeruginosa pneumonia in cystic fibrosis. Thorax. 2010;65(1):57-62.

758 Epub 2009/10/23. doi: 10.1136/thx.2009.114512. PubMed PMID: 19846469.

759 53. Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, et al. Novel

760 cell death program leads to neutrophil extracellular traps. J Cell Biol.

761 2007;176(2):231-41. doi: 10.1083/jcb.200606027. PubMed PMID: 17210947;


763 54. Kolaczkowska E, Jenne CN, Surewaard BG, Thanabalasuriar A, Lee WY,

764 Sanz MJ, et al. Molecular mechanisms of NET formation and degradation revealed by

765 intravital imaging in the liver vasculature. Nature communications. 2015;6:6673.

766 Epub 2015/03/27. doi: 10.1038/ncomms7673. PubMed PMID: 25809117; PubMed


768 55. Lethem MI, James SL, Marriott C, Burke JF. The origin of DNA associated

769 with mucus glycoproteins in cystic fibrosis sputum. Eur Respir J. 1990;3(1):19-23.


771 56. Manzenreiter R, Kienberger F, Marcos V, Schilcher K, Krautgartner WD,

772 Obermayer A, et al. Ultrastructural characterization of cystic fibrosis sputum using

773 atomic force and scanning electron microscopy. Journal of Cystic Fibrosis.

774 2012;11(2):84-92. doi: 10.1016/j.jcf.2011.09.008.



https://doi.org/10.1101/689067

32

775 57. Ramphal R, Lhermitte M, Filliat M, Roussel P. The binding of anti-

776 pseudomonal antibiotics to macromolecules from cystic fibrosis sputum. J Antimicrob

777 Chemother. 1988;22(4):483-90. Epub 1988/10/01. PubMed PMID: 3060459.

778 58. Potter JL, Matthews LW, Spector S, Lemm J. Complex formation between

779 basic antibiotics and deoxyribonucleic acid in human pulmonary secretions.

780 Pediatrics. 1965;36(5):714-20. Epub 1965/11/01. PubMed PMID: 5844004.

781 59. Hunt BE, Weber A, Berger A, Ramsey B, Smith AL. Macromolecular

782 mechanisms of sputum inhibition of tobramycin activity. Antimicrob Agents

783 Chemother. 1995;39(1):34-9. Epub 1995/01/01. PubMed PMID: 7535039; PubMed


785 60. Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human

786 DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci U S A.

787 1990;87(23):9188-92. PubMed PMID: 2251263.

788 61. Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A. Neutrophil

789 elastase targets virulence factors of enterobacteria. Nature. 2002;417(6884):91-4. doi:

790 10.1038/417091a. PubMed PMID: 12018205.

791 62. Janoff A, White R, Carp H, Harel S, Dearing R, Lee D. Lung injury induced

792 by leukocytic proteases. The American journal of pathology. 1979;97(1):111-36.


794 63. Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, et al.

795 Mice lacking neutrophil elastase reveal impaired host defense against gram negative

796 bacterial sepsis. Nat Med. 1998;4(5):615-8. PubMed PMID: 9585238.

797 64. Eggers CT, Murray IA, Delmar VA, Day AG, Craik CS. The periplasmic

798 serine protease inhibitor ecotin protects bacteria against neutrophil elastase. Biochem



https://doi.org/10.1101/689067

33

799 J. 2004;379(Pt 1):107-18. doi: 10.1042/BJ20031790. PubMed PMID: 14705961;


801 65. Floyd M, Winn M, Cullen C, Sil P, Chassaing B, Yoo DG, et al. Swimming

802 Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by

803 Flagellated Pseudomonas aeruginosa. PLoS pathogens. 2016;12(11):e1005987. doi:

804 10.1371/journal.ppat.1005987. PubMed PMID: 27855208; PubMed Central PMCID:

805 PMCPMC5113990.

806 66. Ahmed K, Dai TC, Ichinose A, Masaki H, Nagatake T, Matsumoto K.

807 Neutrophil response to Pseudomonas aeruginosa in respiratory infection. Microbiol

808 Immunol. 1993;37(7):523-9. PubMed PMID: 7694053.

809 67. Lopez-Boado YS, Espinola M, Bahr S, Belaaouaj A. Neutrophil serine

810 proteinases cleave bacterial flagellin, abrogating its host response-inducing activity. J

811 Immunol. 2004;172(1):509-15. PubMed PMID: 14688361.

812 68. Chaban B, Hughes HV, Beeby M. The flagellum in bacterial pathogens: For

813 motility and a whole lot more. Semin Cell Dev Biol. 2015;46:91-103. doi:

814 10.1016/j.semcdb.2015.10.032. PubMed PMID: 26541483.

815 69. Thanabalasuriar A, Scott BNV, Peiseler M, Willson ME, Zeng Z, Warrener P,

816 et al. Neutrophil Extracellular Traps Confine Pseudomonas aeruginosa Ocular

817 Biofilms and Restrict Brain Invasion. Cell host & microbe. 2019. Epub 2019/04/02.

818 doi: 10.1016/j.chom.2019.02.007. PubMed PMID: 30930127.

819 70. Köhler T, van Delden C, Curty LK, Hamzehpour MM, Pechere JC.

820 Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell

821 signaling in Pseudomonas aeruginosa. J Bacteriol. 2001;183(18):5213-22. PubMed

822 PMID: 11514502.



https://doi.org/10.1101/689067

34

823 71. Høiby N. Pseudomonas aeruginosa infection in cystic fibrosis. Relationship

824 between mucoid strains of Pseudomonas aeruginosa and the humoral immune

825 response. Acta Pathol Microbiol Scand [B] Microbiol Immunol. 1974;82(4):551-8.


827 72. Bjarnsholt T, van Gennip M, Jakobsen TH, Christensen LD, Jensen PØ,

828 Givskov M. In vitro screens for quorum sensing inhibitors and in vivo confirmation of

829 their effect. Nat Protoc. 2010;5(2):282-93. Epub 2010/02/06. doi:

830 10.1038/nprot.2009.205. PubMed PMID: 20134428.

831 73. Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg

832 EP. The involvement of cell-to-cell signals in the development of a bacterial biofilm.

833 Science. 1998;280(5361):295-8. Epub 1998/05/02. PubMed PMID: 9535661.

834 74. Bjarnsholt T, Jensen PØ, Burmølle M, Hentzer M, Haagensen JA, Hougen

835 HP, et al. Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and

836 polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology.

837 2005;151(Pt 2):373-83. PubMed PMID: 15699188.

838 75. Vong L, Sherman PM, Glogauer M. Quantification and visualization of

839 neutrophil extracellular traps (NETs) from murine bone marrow-derived neutrophils.

840 Methods Mol Biol. 2013;1031:41-50. Epub 2013/07/05. doi: 10.1007/978-1-62703-

841 481-4_5. PubMed PMID: 23824885.

842 76. Brinkmann V, Goosmann C, Kuhn LI, Zychlinsky A. Automatic

843 quantification of in vitro NET formation. Frontiers in immunology. 2012;3:413. Epub

844 2013/01/15. doi: 10.3389/fimmu.2012.00413. PubMed PMID: 23316198; PubMed




https://doi.org/10.1101/689067

35

846 77. Hu Y. Isolation of human and mouse neutrophils ex vivo and in vitro.

847 Methods Mol Biol. 2012;844:101-13. Epub 2012/01/21. doi: 10.1007/978-1-61779-

848 527-5_7. PubMed PMID: 22262437.

849 78. Kirketerp-Møller K, Jensen PØ, Fazli M, Madsen KG, Pedersen J, Moser C, et

850 al. Distribution, Organization, and Ecology of Bacteria in Chronic Wounds. J Clin

851 Microbiol. 2008;46(8):2717-22. doi: 10.1128/jcm.00501-08.

852 79. Manders EMM, Verbeek FJ, Aten JA. Measurement of co-localization of

853 objects in dual-colour confocal images. Journal of Microscopy. 1993;169(3):375-82.

854 doi: doi:10.1111/j.1365-2818.1993.tb03313.x.

855856 Supporting Information Legends

857 Movie 1

858 Interaction between isolated human polymorphonuclear leukocytes (PMNs) and

859 aggregated P. aeruginosa for 35 min. P. aeruginosa was grown for 24h prior to

860 exposure to PMNs. PMNs are bursting, probably due to production of rhamnolipid by

861 P. aeruginosa. P. aeruginosa are tagged with GFP, and dead cells are stained with

862 propidium iodide (PI).

863

864 Fig. S1. TEM images 48h post-insertion in the murine implant model

865 TEM images displaying P. aeruginosa biofilms and matrix material at 48 h post-

866 insertion in the murine implant model. Matrix material is denoted by black arrows.

867 Scale bars: A) 5 m, B) 2 m.

868

869 Fig. S2 in vitro PMA generated NETs and antibody staining



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870 Isolated human PMNs were stimulated with PMA for 3h in vitro and stained with

871 antibodies. A, B) antibody staining against NE (pink) (A is a merged image of both the

872 antibody (pink) and SYTO9 (green) staining, B) is only the antibody staining), C)

873 antibody staining against H3, no pink staining was observed. D, E) antibody staining

874 against citH3 (D is a merged image of both the antibody and SYTO9 staining, E is only

875 the antibody staining). F) antibody staining using isotype IgG as a control. SYTO9 was

876 used as a counterstain (green). NE and citH3 (pink) were associated to DNA (SYTO9),

877 but H3 was not. No pink was imaged in the control (isotype IgG).

878

879 Fig. S3. Immunostaining of unactivated paraffin embedded human PMNs

880 Unstimulated human PMNs in paraffin sections were stained with NE or H3 (pink).

881 The DNA of the PMNs was stained with SYTO9 (green). NE co-localized with the

882 cytoplasma of the PMNs (A, B) and H3 with the nucleus (C, D). A, C) are merged

883 images of both the antibody (pink) and SYTO9 (green) staining, B, D) are only the

884 antibody staining).

885

886 Fig. S4. Controls for immunostaining

887 Controls for immunostaining of paraffin-embedded sections from the murine implant

888 model and cystic fibrosis (CF) lungs. The paraffin-embedded sections were stained

889 either with either isotype control IgG or in the absence of primary antibody. The

890 secondary antibody was conjugated to Alexa fluor 647 (pink). SYTO9 (green) was used

891 as a counterstain. SYTO9 stains DNA in bacteria and eukaryotic cells. B indicates

892 biofilm and arrow heads points to PMNs.

893



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894 Figure Legends

895

896 Fig. 1.

897 A. P. aeruginosa is internalized by PMNs in the murine implant model. Top row:

898 TEM images showing intact and active PMNs containing internalized P. aeruginosa

899 (arrows) 6h post-insertion of a pre-coated silicone implant (Bar: 2 m). Bottom row:

900 internalized bacteria are magnified (Bar: 500 nm).

901

902 B-D Interaction between PMNs and P. aeruginosa in the murine implant model.

903 Pre-coated silicone implants were inserted into the peritoneal cavity of BALB/c mice.

904 The interaction between PMNs and bacteria was imaged by TEM at 6 h (B), 24 h (C),

905 and 48 h (D) post-insertion. Black arrows: bacteria and biofilms, Arrow heads: PMNs

906 (Bar: 5 m).

907

908 E-G Biofilm formed by P. aeruginosa on a silicone implant at 24 h post-insertion.

909 TEM images showing in vivo biofilms at different magnifications. Matrix material

910 (black arrows) can be seen between the bacteria. E) Bar: 2 m; F) Bar: 1 m; and G)

911 Bar: 200 nm.

912

913 Fig. 2. In vivo DNA labeling of PMNs in the murine implant model.

914 PMNs in the murine implant model were labeled with Click-iT®, in which a modified

915 thymidine analogue, EdU (5-ethynyl-2'-deoxyuridine), is incorporated into DNA

916 during active DNA synthesis in murine immune cells in vivo, and hence will label only

917 DNA originating from murine PMNs. DNA is labeled ex vivo with Alexa Fluor® 647

918 (pink) and counterstained with SYTO9 (green) on an implant 24h post-insertion in the



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919 peritoneal cavity. External to the biofilms SYTO9 (green)-stained DNA strings (white

920 arrows) were also observed as well as a few pink DNA strings (white arrowheads). A,

921 D) merged images showing both EDU (pink) and SYTO9 (green) staining. B, E) images

922 showing only the SYTO9 staining. C, F) images showing only the EDU staining. Red

923 square in A indicates magnified area in D-F.

924

925 Fig. 3. The in vivo biofilm lack PMN-derived DNA 24h post-insertion in the murine

926 implant model.






932 peritoneal cavity. Labeling was observed in PMNs (pink) but was absent from biofilms

933 (double headed arrows), suggesting that PMNs are not a source of eDNA in biofilms.

934 A, D) merged images showing both EDU (pink) and SYTO9 (green) staining. B, E)

935 images showing only the SYTO9 staining. C, F) images showing only the EDU

936 staining. Red squares in A indicates magnified area in D-F and G-I.

937

938 Fig. 4. The in vivo biofilm lack PMN-derived DNA 48h post-insertion in the murine

939 implant model.







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945 peritoneal cavity. Labeling was observed in PMNs (pink) but was absent from biofilms

946 (double headed arrows), suggesting that PMNs are not a source of eDNA in biofilms.

947 A, D) merged images showing both EDU (pink) and SYTO9 (green) staining. B, E)

948 images showing only the SYTO9 staining. C, F) images showing only the EDU

949 staining. Red square in A indicates magnified area in D-F.

950

951 Fig. 5. PNA FISH and DAPI staining of CF lung tissue

952 Aggregated P. aeruginosa (stained with PNA FISH (red)) (white arrows)) and eDNA

953 (stained blue with DAPI) in CF lung tissue (A, B) Strings of DNA (B) are indicated by

954 white arrows (Bar: 9 m).

955

956 Fig. 6. citH3 antibody staining of paraffin-embedded sections from the murine

957 implant model

958 CSLM images showing paraffin-embedded sections of silicone implants from the

959 murine implant model 24h post-insertion. The sections were stained with primary

960 antibodies specific for citrullinated histone H3 (citH3). The secondary antibody was

961 conjugated to Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain.

962 SYTO9 stains DNA in bacteria and eukaryotic cells. A, D) merged images of both the

963 antibody (pink) and SYTO9 (green) staining. B, E) is only the SYTO9 staining. C, F)

964 is only the antibody staining. The images represent two implants. The letter B indicates

965 biofilm.

966

967

968



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969 Fig. 7. H3 antibody staining of paraffin-embedded sections from the murine

970 implant model



973 antibodies specific for histone H3 (H3). The secondary antibody was conjugated to

974 Alexa Fluor 647 (pink). SYTO9 (green) was used as a counterstain. SYTO9 stains DNA

975 in bacteria and eukaryotic cells. A, D) merged images of both the antibody (pink) and

976 SYTO9 (green) staining. B, E) is only the SYTO9 staining. C, F) is only the antibody

977 staining. The images represent two implants. The letter B indicates biofilm.

978

979 Fig. 8. NE antibody staining of paraffin-embedded sections from the murine

980 implant model



983 antibodies specific for neutrophil elastase (NE). The secondary antibody was


985 SYTO9 stains DNA in bacteria and eukaryotic cells. A, D) merged images of both the

986 antibody (pink) and SYTO9 (green) staining. B, E) is only the SYTO9 staining. C, F)

987 is only the antibody staining. The images represent two implants. The letter B indicates

988 biofilm.

989

990 Fig. 9. Immunostaining of paraffin-embedded sections from the CF lung tissue

991 CSLM images showing paraffin-embedded sections of CF lung tissue. The sections

992 were stained with primary antibodies specific for citrullinated histone H3 (citH3) A-C).

993 Histone H3 (D-F) or neutrophil elastase (NE) (G-I). The secondary antibody was



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995 SYTO9 stains DNA in bacteria and eukaryotic cells. A, D, G) merged images of both

996 the antibody (pink) and SYTO9 (green) staining. B, E, H) is only the SYTO9 staining.

997 C, F, I) is only the antibody staining. The images represent samples from three different

998 CF lungs. B indicates biofilm.

999 J: Co-localization of biofilm with antibodies in CF lungs shown as Manders’ co-

1000 localization coefficient. “Outside biofilm” are areas containing PMNs and “biofilms”

1001 are ROI defined biofilms. Sample size (n): NE outside biofilms (16), NE biofilms (20),

1002 H3 outside biofilms (18), H3 biofilms (17), citH3 outside biofilms (15), citH3 biofilms

1003 (18). There was significant difference between H3 outside biofilms and H3 biofilms

1004 (p<0.0001) and citH3 outside biofilms and biofilms (p<0.0007). No significant

1005 difference was found between NE outside biofilms and biofilms (p<0.25). p<0.05 was

1006 considered significant.

1007

1008 Fig. 10. Hypothesis for formation of an eDNA shield in chronic bacterial

1009 infections in vivo

1010 Early during biofilm formation, PMNs are able to phagocytose and destroy single

1011 bacteria or very small particles of bacterial cells. As the biofilm develops, bacterial

1012 aggregates evade phagocytosis and induce a necrotic cell death of PMNs. In chronic

1013 bacterial infections PMNs are continuously recruited to the site of biofilms where they

1014 release eDNA via necrosis. This released eDNA does not become incorporated into the

1015 biofilm itself. The PMN-derived layer of eDNA, which constitutes a secondary matrix,

1016 may provide a passive physical shield for the biofilm against cationic antibiotics such

1017 as tobramycin and additional phagocytes.

1018



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The origin of extracellular DNA in bacterial biofilm infections in vivo · 76 biofilm formation and...

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