Accepted Manuscript Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus) Marina Mathew, Courtney Waugh, Kenneth W. Beagley, Peter Timms, Adam Polkinghorne PII: DOI: Reference:

S0145-305X(14)00151-7 http://dx.doi.org/10.1016/j.dci.2014.05.015 DCI 2202

To appear in:

Developmental & Comparative Immunology

Received Date: Revised Date: Accepted Date:

14 April 2014 22 May 2014 22 May 2014

Please cite this article as: Mathew, M., Waugh, C., Beagley, K.W., Timms, P., Polkinghorne, A., Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus), Developmental & Comparative Immunology (2014), doi: http://dx.doi.org/10.1016/j.dci.2014.05.015

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the

2

koala (Phascolarctos cinereus)

3 4 5 Marina Mathew1, Courtney Waugh1, 2, Kenneth W. Beagley1, Peter Timms1, 2 and, Adam Polkinghorne1, 2*

6 7 8

1

9 10 11 12

Institute of Health & Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove 4059, Brisbane, Australia.

2

Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs 4558, QLD, Australia.

13 14

*Corresponding author:- Faculty of Science, Health, Education and Engineering, University

15

of the Sunshine Coast, 90 Sippy Downs Dr, Sippy Downs 4558, QLD, Australia

16 17

Tel.+61 7 54594674; [email protected]

18 19 20 21 22 23 1

24

Abstract

25

The koala (Phascolarctos cinereus) is an iconic Australian marsupial species that is facing

26

many threats to its survival. Chlamydia pecorum infections are a significant contributor to

27

this ongoing decline. A major limiting factor in our ability to manage and control chlamydial

28

disease in koalas is a limited understanding of the koala’s cell-mediated immune response to

29

infections by this bacterial pathogen. To identify immunological markers associated with

30

chlamydial infection and disease in koalas, we used koala-specific Quantitative Real Time

31

PCR (qrtPCR) assays to profile the cytokine responses of Peripheral Blood Mononuclear

32

Cells (PBMCs) collected from 41 koalas with different stages of chlamydial disease. Target

33

cytokines included the principal Th1 (Interferon gamma; IFNγ), Th2 (Interleukin 10; IL10),

34

and pro-inflammatory cytokines (Tumor Necrosis Factor alpha; TNFα). A novel koala-

35

specific IL17A qrtPCR assay was also developed as part of this study to quantitate the gene

36

expression of this Th17 cytokine in koalas. A statistically significant higher IL17A gene

37

expression was observed in animals with current chlamydial disease compared to animals

38

with asymptomatic chlamydial infection. A modest up-regulation of pro-inflammatory

39

cytokines, such as TNFα and IFNγ, was also observed in these animals with signs of current

40

chlamydial disease. IL10 gene expression was not evident in the majority of animals from

41

both groups. Future longitudinal studies are now required to confirm the role played by

42

cytokines in pathology and/or protection against C. pecorum infection in the koala.

43 44

Keywords

45

Koala, Chlamydia, TNFα, IFNγ, IL10, IL17A

46

2

47

1. Introduction

48

Chlamydia pecorum is an obligate intracellular Gram-negative bacterium, which has been

49

associated with infection and disease in a wide range of animal hosts such as cattle, sheep,

50

goats, and the koala (Longbottom and Coulter, 2003; Mohamad and Rodolakis, 2010;

51

Polkinghorne et al., 2013). In the koala, chlamydial disease is a major contributing factor to

52

localised extinctions of populations in Queensland and New South Wales; indeed a recent

53

study indicated that chlamydiosis was a leading cause of admission to a wildlife care hospital

54

in the latter Australian state, second only to trauma (Griffith et al, 2013). The associated

55

disease caused by C. pecorum chronic infections contributes to the morbidity and mortality of

56

the species via blindness, infertility and in severe cases, death (Polkinghorne et al., 2013).

57

These ocular, urinary, and genital tract pathologies somewhat mirror those seen in humans

58

with C. trachomatis infections (Hemsley and Canfield, 1997).

59 60

Studies of the pathogenesis of chlamydial disease in humans and animal models have shown

61

that the host immunological response to chlamydial infection is the major determinant of

62

protection, as well as immunopathology, of the associated disease (Entrican et al., 2004;

63

Loomis and Starnbach, 2002; Mascellino et al., 2011). Even though protection against

64

chlamydial infection has been traditionally associated with CD4+ mediated pro-inflammatory

65

Th1 response concurrent with IFNγ secretion (Loomis and Starnbach, 2002; Hafner et al.,

66

2008), recent studies in animal models have implicated the IL17A cytokine, secreted by the

67

Th17 cell lineage, to have a similar role through neutrophil recruitment (Scurlock et al., 2011;

68

Andrew et al., 2013). The IL17A cytokine has been shown to play a role in the immune

69

response to extracellular bacteria (Ye, 2001) as well as intracellular bacteria (Lin, 2009).

70

Being a biphasic bacteria, the potential role for the IL17A cytokine in chlamydial immunity

3

71

has generated considerable interest. Studies involving respiratory (Zhou, 2009; Zhang, 2009;

72

Bai, 2009; O’Meara et al., 2013a) and genital tract models of C. muridarum infection in mice

73

(Scurlock, 2011; O’Meara et al., 2013b) have found IL17A production to be protective in the

74

early stages of disease. Further, a predominantly Th2 response, dominated by anti-

75

inflammatory IL10 secretion (Yang, 2001), has been implicated with the fibrotic reaction

76

seen in chlamydial infection due to its suppressive effect on the Th1 response (Miguel et al.,

77

2013).

78 79

We have previously begun to evaluate the IFNγ, IL10 and TNFα cytokine profiles of koalas

80

with current chlamydial disease versus a previous infection (Mathew et al., 2013a; Mathew et

81

al., 2013b). This analysis revealed a strong systemic response in animals with signs of current

82

chlamydial disease. While general trends could be observed, the overall cytokine expression

83

patterns were highly variable across groups, an observation that is not surprising given that:

84

1) the animals were from an out-bred population; 2) the level of disease expression varied;

85

and 3) the time course of infection for each animal was unknown.

86 87

Previous studies conducted in this field did not analyse the role played by IL17A in koala

88

chlamydial disease due to an absence of sequence information and assays (Mathew et al.,

89

2013a; Mathew et al., 2013b). Hence, the current study not only aims to investigate the role

90

played by pro-inflammatory IL17A cytokine in koala chlamydiosis but also aims to analyse a

91

larger cohort of koalas to identify cytokine profiles that may be associated with protective

92

versus disease-associated immune responses. The identification of these koala immunological

93

profiles will help us understand why some animals experience only asymptomatic infection

94

prior to clearance, versus those that develop debilitating immunopathology.

4

95

2.0 Materials and Methods

96

2.1 Ethics statement

97

Queensland University of Technology (QUT) Animal Ethics Committee (Approval No.

98

0700000845) approved the collection and subsequent analysis of the koala blood and swab

99

samples. Blood samples were collected by qualified veterinarians from wild-caught koalas

100

captured as part of a larger field study in the Moreton Bay region of South East Queensland,

101

Australia. Samples were collected from animals anaesthetised as part of a thorough health

102

evaluation.

103 104 105 106

2.2 Sample Collection

107

and stored at 4oC for processing within 24 hours of collection. Swabs were collected from the

108

conjunctiva of the left eye and right eye, urogenital sinus (females) and urethra (males) using

109

aluminium shafted cotton tipped swabs (Copan, Interpath Services, Melbourne).

Blood samples (5-6 mL) from 41 wild-caught anaesthetised koalas, were collected in EDTA

110 111

2.3 Koala lymphocyte proliferation, RNA extraction and reverse transcription assays

112

Koala peripheral blood mononuclear cells (PBMCs) were harvested from the blood samples

113

and suspended to a final concentration of 2 x 106 cells/ml in RPMI 1640 T cell media, as

114

previously described (Mathew et al., 2013a). Cells mixed with equal volume of T cell

115

mitogens PMA (1µg/ml) and Ionomycin (50ng/ml) were used as positive controls for

116

lymphocyte proliferation. Koala PBMCs were stimulated with UV-inactivated C. pecorum G

117

at 1/20 dilution to estimate cytokine production upon exposure to chlamydial antigens. RNA

118

extraction and cDNA synthesis were completed from cells harvested at 0, 12 and 24 hours

119

post-stimulation as described in Mathew et al., 2013a. 5

120

2.4 Bioinformatic analysis

121

Multiple sequence alignments were constructed using the Geneious Alignment program in

122

the Geneious Pro 5.6.5 software and GeneDoc version 2.7.000 software. Predicted marsupial

123

IL17A sequences were obtained from Ensembl: opossum (ENSMODT00000023892),

124

Tasmanian devil (ENSSHAT00000014284) and wallaby (ENSMEUT0000005320).

125 126

2.5 Cloning and sequence analysis of Koala IL17A mRNA sequence

127

Primers were designed based on a homologous sequence alignment constructed using IL17A

128

sequence from other marsupial species including the wallaby, opossum and Tasmanian devil.

129

Using wallaby IL17A sequence as the reference, primers were designed targeting a 450bp

130

sequence of the coding region. Conventional PCR was performed on cDNA samples pooled

131

together from PBMCs stimulated with PMA/Ionomycin at 12 and 24 hours, and unstimulated

132

samples at 0 hours. After PCR amplification of a product of the expected size, this was

133

cloned into a plasmid using the Promega pGEM-T Easy Vector Systems I as previously

134

described (Mathew et al., 2013a) and sequenced at Australian Genome Research Facility

135

using the AB 3730xl platform.

136 137

2.6 IL17A qrtPCR assay design and optimisation

138

The koala IL17A sequence identified was utilised to develop a qrtPCR assay with SYBR

139

green I dye based chemistry to amplify a 282bp product of the koala IL17A coding sequence.

140

Primer

141

GGAGTCTCAATGCCAAAGAGG,

sequences

for

this

reaction

are

as

follows:

IL17Af

-

IL17Ar - GACGGAGTTCACGTGGTGGT. The

6

142

primer pairs were designed to amplify a PCR product that spanned over two exons in order to

143

avoid co-amplification of genomic DNA in the PCR reaction.

144 145

A standard curve was constructed using serial dilutions with known concentrations of the

146

282bp PCR product to determine assay efficiency as previously described (Mathew et al.,

147

2013a). Reactions were carried out in a Corbett Rotor Gene 6000 real time PCR machine at a

148

final volume of 20 µl, with 1 unit of FastStart Taq Polymerase (Roche), 2 µl of 25mM MgCl2

149

(Roche), 2 µl of 10 x buffer (Roche), 0.6 µl of 10 mm dNTPs (Roche), 3 µl of 1/10000

150

SYBR Green, 0.6 µl each of 10 mM forward and reverse primers each and 2 µl of template

151

DNA. After an initial incubation of 95oC for 10 mins, 40 cycles of 20 s at 95 oC, 25 s at 65oC

152

and 25 s of 72oC were carried out for each IL17A qrtPCR. All samples were tested in

153

duplicate. A mastermix with no cDNA was used as the no template control, and water was

154

used as the negative control.

155 156

A glyceraldehyde 3-phosphate dehydrogenase (GAPDH) qrtPCR assay described in Mathew

157

et al. (2013a) was used as a reference gene for relative quantification. GAPDH has been

158

chosen as the reference gene because of its relatively stable expression following stimulation

159

with the mitogens as well as the test antigen used in this study. This finding has been

160

confirmed by Maher et al. (2014) who reported GAPDH to be a suitable housekeeping gene

161

for normalising cytokine expression by koala lymphocytes. The IL17A cytokine gene was

162

normalised to GAPDH by the 2 -∆∆CT method, where ∆∆CT = (Ct of target - Ct of GAPDH) at

163

any time point - (Ct of target - Ct of GAPDH) at time zero hour (Livak and Schmittgen,

164

2001).

7

165 166

2.7 Cytokine targets for analysis

167

IFNγ, IL10, TNFα, and GAPDH mRNA expression levels were determined using optimised

168

koala-specific qrtPCR assays, as previously reported (Mathew et al., 2013a; Mathew et al.,

169

2013b). The qrtPCR primers used in this study are summarised in Table 1. Prior to this study,

170

the koala IL17A sequence was not known and so no IL17A assay was available.

171 172

2.8 C. pecorum infection status via PCR and western blot

173

The koalas captured as part of this study were screened for the presence of current or

174

previous C. pecorum infection via two approaches: 1) 16SrRNA C. pecorum species-specific

175

qrtPCR assays applied to swab samples collected from the conjunctiva and urogenital sinuses

176

of koalas (Wan et al., 2011); and 2) for the presence of Chlamydia specific antibodies in

177

plasma using C. pecorum His-tagged major outer membrane protein (MOMP) A, F and G

178

western blot screening (Kollipara et al., 2012).

179

2.9 Statistical analysis

180

Mann Whitney U test with a P value set at 0.05 was used to analyse the significance of target

181

cytokine gene expression in Group I/ No Disease vs Group II/ Diseased with reference to

182

GAPDH. Statistical comparison between different cytokine gene expressions within a group

183

was performed using the Wilcoxon matched pair test. Graph-Pad Prism version 5 (Graph Pad

184

Software, Lajolla, CA, USA) was used to perform statistical analyses.

185 186

3.0 Results

8

187

3.1 Koala IL17A nucleotide sequence identification

188

Prior to the commencement of this study, no sequence or assays were available for measuring

189

the koala IL17A immune response. For this purpose, we cloned and sequenced the coding

190

region of koala IL17A. Using primers designed to conserved regions of the predicted

191

opossum, wallaby and Tasmanian devil IL17A sequences, a 462 base pair partial IL17A

192

mRNA sequence was identified following PCR amplification and sequencing (GenBank

193

under Accession number KJ174517). Not surprisingly, similar to the sequences for other

194

koala cytokines that we recently reported (Mathew et al, 2013a & b), koala IL17A shares

195

greatest similarity to the algorithm-predicted gene sequences for marsupial IL17A sequences

196

of

197

ENSMODT00000023892) and Tasmanian devil (83.8%; ENSSHAT00000014284) compared

198

to experimentally-verified mammalian (e.g. Human –69%; GenBank ID - BC067505) or

199

avian sequences (e.g. Chicken – 51.7%; GenBank ID - AM773756).

the

tammar

wallaby

(89.4%;

ENSMEUT0000005320),

opossum

(84.2%;

200 201

3.2 IL17A qrtPCR assay development and optimisation

202

A koala-specific IL17A qrtPCR was developed to measure IL17A gene expression in the

203

peripheral blood of the koala. The primers were designed to span over at least one intron-

204

exon junction to avoid co-amplification of any residual genomic DNA. IL17A gene

205

expression was measured at 0, 12, and 24 hours in: 1) unstimulated koala PBMCs; 2) PBMCs

206

stimulated with PMA/ Ionomycin; and 3) PBMCs stimulated with UV-inactivated C.

207

pecorum strain G. In many of the animals there was no observable IL17A gene expression in

208

unstimulated PBMCs at 0 hours. For the purpose of analysis, these samples were assigned to

209

have a Ct value of 30. The reaction has a detection limit of approximately 2 copies at a

210

threshold of approximately 30 cycles (data not shown). Hence, the samples with no

9

211

detectable IL17A at 0 hours in unstimulated samples were assigned a Ct of 30 to enable use

212

of the 2 -∆∆CT method and as such enabling comparison of expression levels amongst the

213

different cytokines.

214

Following assay optimization, an R2 value of 0.99 and reaction efficiency of greater than

215

90% was observed in all subsequent PCR runs. Reproducibility of the assay was measured

216

and confirmed by comparing the Ct values of standards in consecutive runs, which were

217

found to be similar. These parameters illustrated that the IL17A qrtPCR assay was optimised

218

and could be used for quantification of this gene in an unknown sample.

219 220

3.3 Characterisation of the current and previous Chlamydia infection status of

221

asymptomatically infected and diseased koala cohorts

222

Forty one koalas were captured, clinically examined and sampled as a part of an ongoing

223

study of healthy koalas in a wild population in South-East Queensland. At the time of

224

sampling, 12 of these animals displayed evidence of chlamydial disease (Group II; Table 3),

225

with 11 animals presenting with cystitis only with no evidence of ocular disease, while the

226

remaining one animal had conjunctivitis only. The remaining 29 animals displayed no signs

227

of chlamydiosis (Group I; Table 2).

228

To investigate their current or previous chlamydial infection status, C. pecorum species-

229

specific qPCR assays and/or anti-C. pecorum MOMP Western blots were performed (Table 2

230

and 3). Initially, the animals were screened using the 16S rRNA qrtPCR. The qrtPCR

231

negative animals were then also screened by MOMP western blot. Of the 41 animals studied,

232

only three animals were negative for C. pecorum 16S rRNA, however, these animals tested

233

positive for MOMP antibody via Western blot screening, indicating that they had seen a

10

234

previous infection. Based on these results and the presence of clinical signs of chlamydiosis,

235

the 41 koalas were grouped as follows: Group I/ No Disease (n = 29) – C. pecorum PCR

236

and/or western blot positive without overt signs of chlamydiosis; Group II/ Diseased (n = 12)

237

– koalas with current signs of chlamydiosis and C. pecorum PCR positive. Evaluation of the

238

presence or absence of clinical signs of chlamydiosis was performed by experienced

239

veterinarians from Endeavour Veterinary Ecology, (EVE) Toorbul, Australia. Group II/

240

Diseased animals were further classified into koalas with active (n=9) and inactive (n=3)

241

disease based on the veterinary evaluation and chlamydial disease scoring criteria

242

recommended by Wan et al. (2011). The presence of pyuria and suppurative exudate at the

243

ocular and urogenital site was considered to be characteristic of active chlamydial disease.

244

The presence of inflammatory cells in the urine sediment of koalas with cystitis was a further

245

indication of active disease. Inactive chlamydial disease was classified as disease in the

246

absence of these additional features.

247 248

Of the 29 animals included in Group I/No Disease, 26/29 of the animals in this group were

249

PCR positive for C. pecorum DNA. For all but one of these PCR positive animals, C.

250

pecorum DNA could be detected at both the UGT and at least one of the two ocular sites. The

251

exception to this group of PCR positive animals was one koala (My), who was found to be

252

PCR positive in both eyes but negative for C. pecorum in the UGT. The remaining three

253

animals in this group that were PCR negative, however, were found to be serology positive

254

using our C. pecorum MOMP Western blots (Table 2; data not shown).

255

For the 12 animals in Group II/Diseased (Table 3), all animals were found to be PCR positive

256

in the UGT. Ocular shedding was less common, however, with C. pecorum DNA detected in

257

only one animal with cystitis (Barry) and in an animal (Beauty) with signs of unilateral

258

conjunctivitis in the right eye (Table 3). 11

259

3.4 Expression profiling of the IFNγ, IL10, IL17A and TNFα responses of Chlamydia-infected

260

koalas with or without clinical disease

261 262

In order to evaluate the immune-recognition and systemic cytokine response of koalas with

263

current or previous C. pecorum infection, PBMCs isolated from the koalas were stimulated

264

with UV inactivated, semi-purified C. pecorum G for 12 and 24 hours. IL17A, TNFα, IFNγ,

265

and IL10 gene expression was measured as previously described (Mathew et al., 2013a;

266

Mathew et al., 2013b). With one as the base line for unstimulated PBMCs at 0 hour, fold

267

increase in gene expression was measured for each cytokine of interest.

268

Duplicate stimulation experiments were performed with PMA/ Ionomycin stimulation to

269

evaluate cell viability and to generate positive controls for our qrtPCRs. PMA/Ionomycin has

270

been found to be an effective mitogen to stimulate koala PBMCs in a recent study (Maher et

271

al., 2014). Strong expression, defined as a > 50 fold increase in expression following

272

stimulation, could be observed in at least one cytokine (TNFα, IFNγ and IL17A) for the

273

majority of koalas (38/41, 92%). IL10 expression, on the other hand was lower compared to

274

the other cytokines across both cohorts, with an average fold change < 10 for all but four of

275

the 41 animals. The latter four animals also expressed high levels of one of the other three

276

cytokines.

277 278

When PBMCs from Group I/ No Disease (n=29) animals with evidence of current or past C.

279

pecorum infection in the absence of chlamydial disease were stimulated with UV inactivated

280

C. pecorum G, significant variation in the cytokine responses were observed. The geometric

281

mean, median and range of the four cytokines quantified for the animals in this cohort is

282

summarised in Table 4. Of the 29 animals analysed in Group I/ No Disease, a very low

12

283

IL17A gene expression ranging between 0 – 1 was observed in 16 animals (55%) following

284

exposure to C. pecorum. Koalas which had a gene expression of 0 for IL17A, were assigned

285

the fold increase as 0.01, which is the lowest detectable fold increase in the cohort of koalas

286

included in this study. For 11 of the 29 koalas, IL17A transcripts could only be detected

287

following antigen stimulation and not at the 0 hour time point. Seven of the 29 koalas (24%)

288

koalas did not have any detectable IL17A transcription within 24 hours of C. pecorum

289

stimulation.

290 291

Amongst the other cytokines analysed, the lowest fold change increase was observed for

292

IFNγ gene expression with a geometric mean of 2.1 and fold change ranging between 0.02 –

293

364.6. When comparing the expression profiles of each of the cytokines in this group, TNFα

294

gene expression was found to be significantly higher (p<0.05) than the expression of IFNγ

295

and IL10. There were no statistically significant difference in the IL17A expression within

296

Group I/ No Disease.

297 298

Similar to the Group I/No Disease animals, there was considerable animal-to-animal

299

variation in the cytokine responses of Group II/Diseased animals with signs of current

300

chlamydial disease and evidence of an ongoing C. pecorum infection (n=12), however, some

301

trends could be observed (Table 4). The strongest expression results for animals in this group

302

following C. pecorum antigen stimulation were for IL17A, with a geometric mean of 34.8

303

and fold change range of 1.4 - 445. A relatively lower IL17A expression was observed only

304

for one animal with conjunctivitis (2.0 fold change) and another two animals whose

305

expression response for all the tested cytokines was relatively low. The observed fold change

306

in gene expression for IL17A was significantly higher than that observed for IL10 (p<0.03)

13

307

and TNFα as well (p<0.01). Though not statistically significant, a general trend of higher

308

IL17A gene expression was observed in koalas with active chlamydial urogenital disease

309

than in animals with inactive disease.

310 311

When comparing the gene expression profiles of the two cohorts together, IL17A expression

312

was found to be significantly “stronger” in the Group II/Diseased group animals (p<0.009).

313

There were otherwise no statistically significant differences between the cytokine gene

314

expressions of both groups, although a general trend towards proinflammatory cytokine

315

secretion was observed in the Group II/Diseased animals (Table 4). Hence, the key difference

316

in the cytokine profile of the two groups was a statistically significant IL17A gene expression

317

amongst the diseased group of animals.

318 319 320

Discussion

321

Chlamydia is a bacterium with a complex biphasic development cycle, involving inter-

322

conversion between extracellular and intracellular forms. It has been hypothesised that the

323

response to both these intracellular and extracellular developmental forms requires a balanced

324

Th1 and Th2 response (Darville and Hiltke, 2010). Several recent studies have also suggested

325

a seminal role for Th17 in chlamydial pathogenesis (Andrew et al., 2013; O’Meara et al.,

326

2013a; O’Meara 2013b). However, extensive studies in humans are yet to truly understand

327

the natural immune response to this obligate intracellular bacterium (Geisler, 2010), let alone

328

the differences that will influence a protective versus pathological outcome (Agrawal et al.,

329

2009).

14

330 331

In the current study, we performed a cross-sectional profiling of Chlamydia-infected koalas

332

with and without evidence of chlamydial disease by measurement of a range of important

333

cytokine gene markers. Though not statistically significant, a trend towards a higher pro-

334

inflammatory cytokine profile was observed in Group II/Diseased animals when compared to

335

the Group I/No Disease animals. Although the immune response of animals within each

336

cohort was highly variable, significant differences were observed in the IL17A response of

337

koalas in Group I/No Disease and Group II/ Diseased, with animals in the latter group

338

showing strong increases in IL17A gene expression following exposure to C. pecorum

339

antigen. Although we do not have cell-specific markers for koalas, the strong fold-change in

340

expression of these koalas suggests that diseased koalas have high levels of Th17 cells in

341

their peripheral blood. In other animal models, αβ T cells, γδ T cells, invariant Natural Killer

342

T cells (iNKT) cells, Lymphoid Tissue inducer (LTi)-like cells, natural killer cells and

343

macrophages are also known to produce IL17A (Cua and Tato, 2010; Jin and Dong, 2013). In

344

animal models of chlamydial infection, IL17A has been shown to aid Th1 immunity

345

development and neutrophil recruitment (Scurlock et al., 2011).

346 347

IL17 and IFNγ have been shown to have a synergistic effect on the up regulation of iNOS

348

and NO production (Zhang et al., 2012), however, they appear to have opposing functions

349

when it comes to neutrophil recruitment (Savarin et al., 2012). Similar to IFNγ, nevertheless,

350

IL17A has also been implicated in immunopathology, with evidence observed of increased

351

recruitment of inflammatory cells such as neutrophils and macrophages and increased

352

expression of matrix metaloproteinases at the site of infection (Andrew et al, 2013; O’Meara

353

et al., 2013b). While we have no direct ability to currently assess these inflammatory factors

354

in the koala, the relatively higher IL17A gene expression observed predominantly in animals

15

355

with active chlamydial disease, evidenced by suppurative exudation and inflammatory cells

356

in urine sediment, as opposed to inactive disease in Group II/Diseased animals (Table 3)

357

could be consistent with this “over-amplification” of the inflammatory response to the

358

presence of the infection, especially in light of the low expression of IFNγ and IL10 in these

359

animals.

360 361

Observations similar to our study have been reported by Jha et al. (2011) who showed greater

362

expression of IL17A in comparison to IFNγ in the cervical washes of women with C.

363

trachomatis cervicitis. Contrary observations have also been described (Barral et al., 2014)

364

which can be attributed to the differences in the study population; the former looked at cases

365

of cervicitis whereas the latter focused on asymptomatic infection. However, vaccination has

366

been reported to be less effective in the absence of IL17A in a mouse model of C. muridarum

367

genital tract infection (Andrew et al., 2013). Similarly, IFNγ/ IL17A double positive CD4+ T

368

cells correlated with improved protection against chlamydial infection in a vaccine study (Yu

369

et al., 2010). While clearly more studies are needed to determine the role of IL17A in

370

chlamydial disease pathology, our study does indicate an important role for IL17A gene

371

expression in chlamydial disease development in the koala.

372

Similar to our previous studies (Mathew et al., 2013a; Mathew et al., 2013b), a comparatively

373

strong expression of TNFα and IFNγ, normally associated with protection, was observed in

374

animals with current signs of chlamydiosis (Group II) and not in animals without any sign of

375

disease (Group I). This is not surprising as the very immune responses which are associated

376

with protection against members of the genus Chlamydia have also been implicated in

377

disease pathology (Loomis and Starnbach, 2002). To explain this phenomenon, it has been

378

hypothesized that the possibility of a persistent phase of chlamydial infection (Hogan et al.,

379

2004) and the biphasic life cycle of the organism provides avenues to escape the pro-

16

380

inflammatory immune response of the host and to set in motion a chain of intermittent Th1

381

and Th2 immune responses through the course of its developmental cycle, eventually leading

382

to the tissue damage and irreversible sequelae associated with chronic chlamydial disease as

383

observed in the Group II/Diseased animals in this study (Mascellino et al., 2011). However,

384

IL10, which has been noted to mitigate the damaging effects of the pro-inflammatory

385

response was at low expression in both the groups analysed in our koala study. This could be

386

due to fewer IL10 producing cells in the peripheral circulation as a low IL10 expression was

387

also noted following mitogen stimulation in all the studied animals.

388 389

Marked variations in cytokine response could also be observed in animals included in Group

390

I/No Disease and Group II/Diseased, as previously observed (Mathew et al., 2013a; Mathew

391

et al., 2013b), despite the increase in the cohort sizes in this study. Again, this is not

392

surprising as the koalas were likely to have been infected at variable points of time and could

393

essentially be at different stages of infection. While logistically challenging in naturally

394

infected wild animals, longitudinal studies involving assessment of koala infection and health

395

status will need to be undertaken to understand the exact role that these cytokines have on

396

infection and disease progression. Controlling for the fact that koalas are an outbred

397

population will be more challenging. In humans, genetic variability in the HLA class I and II

398

genes (Geisler et al., 2004) and IL10 promoter gene polymorphisms (Wang et al., 2005) have

399

been linked to increased susceptibility or protection against C. trachomatis infection.

400

Targeting island koala populations with lower genetic diversity (Lee et al., 2013) in future

401

studies can help control for the genetic variation observed in outbred koala populations;

402

thereby helping to understand its contribution to the variable immune response observed.

17

403

Beyond further studies to delineate the populations of immune cells in systemic circulation

404

that may be involved in the koala response to chlamydial infection, it will also be important

405

to understand local immune responses to this mucosal pathogen. It has been previously

406

demonstrated that significant variation exists in the in vitro immune response elicited by

407

PBMCs when compared to lymphocytes from the site of actual infection during C.

408

trachomatis infection (Vats et al., 2007).

409

infection will help in better understanding the immune response of koala chlamydial

410

infection. Future studies would also benefit from incorporation of age and sex matched

411

control animals without history of previous chlamydial infection; however obtaining

412

uninfected wild animals remains a major challenge. Another variable that will need to be

413

considered in these studies includes (a) the effect of anaesthetics, known to influence

414

cytokine expression pattern following in-vitro mitogen stimulation of lymphocytes in humans

415

(Schneemilch et al., 2004), or (b) physiological stress (Abraham, 1991), on koala cytokine

416

gene expression as little or nothing is known about either for this native species.

Hence, assays targeting the mucosal site of

417 418

This study has provided us with important baseline data that will assist in understanding

419

chlamydial disease pathogenesis and protection, a key consideration for the development of a

420

safe and effective chlamydial vaccine. The koala specific immunological tools developed in

421

this study for IL17A, alongside our previously described TNFα, IFNγ and IL10 assays, will

422

be critical for the subsequent work but may also eventually form a part of a immunological

423

“toolkit” (Maher et al., 2014; Morris et al., 2014) that could be available to stakeholders to

424

assess the health status and impact of chlamydial infections in affected koala populations.

425 426

18

427

Acknowledgements

428

We thank the veterinarians at the Endeavour Veterinary Ecology, (EVE) Toorbul, Australia

429

for helping with koala blood sample collection. This work was financially supported by an

430

ARC Linkage Grant awarded to PT, KB and AP and a Queensland Government NIRAP

431

Scheme Grant.

432 433 434

19

435

Table 1. qrtPCR primers for TNFα, IFNγ, IL17A, IL10 and GAPDH Gene

Forward Primer 5’-3’

Reverse Primer 5’-3’

Target

TNFα

GAGACGTAGAGCTAGCAG

TGCCAAGAAAATCTGTGGAC

180bp

IFNγ

AGCTACCTCTTAGCATCC

TCCTCTTTCCAA CGATCC

167bp

IL17A

GGAGTCTCAATGCCAAAGAGG

GACGGAGTTCACGTGGTGGT

282bp

IL10

TGGGCTCTTTAGGCGAGAAG

CAGGGCAGGAATCTGTGACA

72bp

GAPDH

GGACTCATGACCACAGT

CCATCACGCCACAGC

70bp

436 437 438 439 440 441 442 443

20

444

Table 2. PCR, Western Blot and cytokine gene results for animals included in the Group I/

445

No disease in this study.

446

16S qPCR LE RE UGT Mel G NEG NEG NEG Slocombe NEG POS POS Caz NEG NEG NEG Julia NEG POS POS Lexi NEG NEG NEG My POS POS NEG Cindy NEG NEG POS Nat NEG POS POS Sarah NEG NEG POS Kate G NEG POS POS Doddy POS POS POS Susan POS POS POS Cougar NEG NEG POS Karen NEG NEG POS Matt NEG NEG POS Lindsay NEG NEG POS Cate NEG NEG POS Bubbles NEG NEG POS Tash POS POS POS Gav POS POS POS Bev POS POS POS Salvatore NEG POS POS Frances POS POS POS Mark POS POS POS Daryl NEG NEG POS Robyn NEG NEG POS Mango NEG NEG POS Minky NEG NEG POS Coco NEG NEG POS LE - Left Eye, RE – Right Eye, Animal

Western Blot A F G TNFα NEG POS NEG 151 101.8 NEG NEG POS 4 0.9 NEG NEG POS 24.5 112.2 88 5.2 27.6 36.2 270.5 116.1 93.7 107.6 11.3 18.1 7.9 1.5 3.2 0.1 15.8 1.9 0.3 1.3 1.4 20.9 10.1 3.5 0.9 NEG- negative, POS- positive. A,

Fold increase IFNγ IL17A IL10 35.7 0.01 6.7 1.2 200.8 33.5 0.4 0.01 4.9 0.5 71.5 5.3 53.4 162 2.0 199.4 0.01 38.0 3.9 71 8.7 1.3 238.8 24.4 0.6 0.01 8.6 364.5 15.6 7.9 9.2 0.01 2.7 0.1 0.6 0.2 0.9 10.3 9.0 14.8 2.1 11.7 0.5 337.7 5.0 0.7 0.2 4.8 1.2 0.01 10.3 49.1 0.8 21.1 1.6 14.8 2.4 1.1 0.01 1.5 0.5 0.4 2.3 3.2 0.01 19.9 0.2 165.4 3.7 1.3 0.01 0.5 3.5 0.01 1.2 0.5 4.9 3.9 2.6 22.8 14 0.5 0.1 2.8 0.02 0.01 2.3 F and G are three C. pecorum

447

MOMP (Major Outer Membrane Protein) aminotypes described in eastern Australia by Kollipara

448

et al (2013). POS/NEG by 16S qPCR indicates the presence/absence of C. pecorum DNA at the

449

site. POS/NEG by western blot for A, F and G aminotypes indicates presence of specific

450

antibodies to these strains in the sera of the koala. ‘-’ sign indicates animals for which western

451

blot was not done when they were positive by 16SrRNA qPCR. The fold increase in gene

452

expression is calculated by 2-∆∆CT method as described in section 2.6.

453

21

454 455 456

Table 3. 16S qPCR and cytokine gene results for animals included in the Group II/ Diseased in this study. Animal

Disease

Curry Poma

Chronic active cystitis Chronic active cystitis Subclinical active cystitis Chronic active cystitis Chronic active cystitis Acute active cystitis Subclinical active cystitis Chronic active cystitis Chronic inactive cystitis Chronic inactive cystitis Sub-acute inactive cystitis Chronic active conjunctivitis

Mali Delores Circe Barry Harley KP Rambo Pinki Andrew Beauty

LE NEG NEG

16S qPCR RE UGT NEG POS NEG POS

TNFα 25.9 14.6

Fold increase IFNγ IL17A 1.2 153.2 0.0 286

IL10 32.6 2.3

NEG

NEG

POS

2.2

3.7

1.3

2.8

NEG NEG NEG

NEG NEG POS

POS POS POS

35.2 32.4 32

2.0 831.7 12.9

445.7 137.1 103.2

2.8 1.2 129.7

NEG

NEG

POS

8.8

33.1

63.1

0.5

NEG NEG NEG

NEG NEG NEG

POS POS POS

27 13.9 6.3

5.5 56.1 4.0

49.5 16.7 18.6

13.0 15.0 10.3

NEG

NEG

POS

5.9

6.6

4.1

1.2

NEG

POS

POS

45.2

1.0

1.9

16.7

457

22

458

Table 4. Geometric mean, median and range of the four cytokines quantified in the

459

Group I and Group II animals

Number of koalas

Group 1

Group 2

29

12

TNFα -

Geometric mean1

10.1

14.5

-

Median

11.3

14.7

-

Range

0.1 – 270.6

2.5 - 45.3

-

Geometric mean1

2.1

5.8

-

Median

1.3

4.8

-

Range

0.02 - 364.6

0.5 - 86.2

-

Geometric mean2

0.7

34.8

-

Median

0.6

56.3

-

Range

0.01 – 337.8

1.4 – 445.7

-

Geometric mean1

5

6.0

-

Median

5.04

6.6

-

Range

0.3 – 38.1

0.6 – 129.8

IFNγ

IL17

IL10

460 461

1

462

TNFα (p=0.57), IFNγ (p=0.08) and IL10 (p=0.87).

463

2

464

(p=0.009).

No statistically significant difference was observed for expression between Group I vs Group II for

Differences between the IL17 gene expression for Group vs Group II are statistically significant

23

465

References

466

Abraham, E., 1991. Effects of stress on cytokine production. Methods Achiev. Exp.

467

Pathol. 14, 45-62.

468 469

Agrawal, T., Gupta, R., Dutta, R., Srivastava, P., Bhengraj, A.R., Salhan, S., Mittal, A.,

470

2009. Protective or pathogenic immune response to genital chlamydial infection in

471

women - A Possible role of cytokine secretion profile of cervical mucosal cells. Clin.

472

Immunol. 130, 347-354.

473 474

Andrew, D.W., Cochrane, M., Schripsema, J.H., Ramsey, K.H., Dando, S.J., O'Meara,

475

C.P., Timms, P., Beagley, K.W., 2013. The duration of Chlamydia muridarum genital

476

tract infection and associated chronic pathological changes are reduced in IL-17 knockout

477

mice but protection is not increased further by immunization. PLoS ONE. 8(9).

478

http://dx.doi:10.1371/journal.pone.0076664.

479 480

Bai, H., Cheng, J., Gao, X., Joyee, A.G., Fan, Y., Wang, S., Jiao, L., Yao, Z., Yang, X.,

481

2009. IL-17/Th17 promotes type 1 T cell immunity against pulmonary intracellular

482

bacterial infection through modulating dendritic cell function. J. Immunol. 183, 5886-

483

5895. http://dx.doi: 5810.4049/jimmunol.0901584.

484 485

Barral, R., Desai, R., Zheng, X., Frazer, L.C., Sucato, G.S., Haggerty, C.L., O'Connell,

486

C.M., Zurenski, M.A., Darville, T., 2014. Frequency of Chlamydia trachomatis-specific

487

T cell interferon-gamma and interleukin-17 responses in CD4-enriched peripheral blood

488

mononuclear cells of sexually active adolescent females. J. Reprod. Immunol.

489

http://dx.doi:10.1016/j.jri.2014.01.002.

24

490 491

Burton, M.J., Ramadhani, A., Weiss, H.A., Hu, V., Massae, P., Burr, S.E., Shangali, W.,

492

Holland, M.J., Mabey, D.C., Bailey, R.L., 2011. Active trachoma is associated with

493

increased conjunctival expression of IL17A and profibrotic cytokines. Infect. Immun. 79,

494

4977-4983.

495 496

Cua, D.J., Tato, C.M., 2010. Innate IL-17-producing cells: the sentinels of the immune

497

system. Nat. Rev. Immunol. 10, 479-489.

498 499

Darville, T., Hiltke, T.J., 2010. Pathogenesis of genital tract disease due to Chlamydia

500

trachomatis. J. Infect. Dis. 201, 114-125.

501 502

Entrican, G., Wattegedera, S., Rocchi, M., Fleming, D.C., Kelly, R.W., Wathne, G.,

503

Magdalenic, V., Howie, S.E.M., 2004. Induction of inflammatory host immune responses

504

by organisms belonging to the genera Chlamydia/Chlamydophila. Vet. Imm. Immpath.

505

100, 179-186.

506 507

Geisler, W.M., Tang, J., Wang, C., Wilson, C.M., Kaslow, R.A., 2004. Epidemiological

508

and genetic correlates of incident Chlamydia trachomatis infection in North American

509

adolescents. J. Infect. Dis. 190, 1723-1729.

510 511

Geisler, W.M., 2010. Duration of untreated, uncomplicated Chlamydia trachomatis

512

genital infection and factors associated with chlamydia resolution: a review of human

513

studies. J. Infect. Dis. 201 Suppl 2, 104-113.

514

25

515

Griffith, J.E., Dhand, N.K., Krockenberger, M.B., Higgins, D.P., 2013. A retrospective

516

study of admission trends of koalas to a rehabilitation facility over 30 years. J. Wildl. Dis.

517

49, 18-28.

518 519

Hafner, L., Beagley, K., Timms, P., 2008. Chlamydia trachomatis infection: host immune

520

responses and potential vaccines. Mucs. Imm. http://dx.doi: 10.1038/mi.2007.19.

521 522

Hemsley, S., Canfield, P.J., 1997. Histopathological and immunohistochemical

523

investigation

524

inflammation in koalas (Phascolarctos cinereus). J. Comp. Pathol. 116, 273-290.

of

naturally

occurring

chlamydial

conjunctivitis

and

urogenital

525 526

Hogan, R.J., Mathews, S.A., Mukhopadhyay, S., Summersgill, J.T., Timms, P., 2004.

527

Chlamydial persistence: beyond the biphasic paradigm. Infect. Immun. 72, 1843-1855.

528 529

Holland, M.J., Bailey, R.L., Conway, D.J., Culley, F., Miranpuri, G., Byrne, G.I., Whittle,

530

H.C., Mabey, D.C.W., 1996. T helper type-1 (Th1)/Th2 profiles of peripheral blood

531

mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects

532

with severe trachomatous scarring. Clin. Exp. Immunol. 105, 429-435.

533 534

Jha, R., Srivastava, P., Salhan, S., Finckh, A., Gabay, C., Mittal, A., Bas, S., 2011.

535

Spontaneous secretion of interleukin-17 and -22 by human cervical cells in Chlamydia

536

trachomatis infection. Microbes Infect. 13, 167-178.

537 538

Jin, W., Dong, C., 2013. IL-17 cytokines in immunity and inflammation. Emerg.

539

Microbes. Infect. 2, http://dx.doi. 10.1038/emi.2013.58.

26

540 541

Kollipara, A., George, C., Hanger, J., Loader, J., Polkinghorne, A., Beagley, K., Timms,

542

P., 2012. Vaccination of healthy and diseased koalas (Phascolarctos cinereus) with a

543

Chlamydia pecorum multi-subunit vaccine: Evaluation of immunity and pathology.

544

Vaccine 30, 1875-1885.

545 546

Kollipara, A., Polkinghorne, A., Wan, C., Kanyoka, P., Hanger, J., Loader, J., Callaghan,

547

J., Bell, A., Ellis, W., Fitzgibbon, S., Melzer, A., Beagley, K., Timms, P., 2013. Genetic

548

diversity of Chlamydia pecorum strains in wild koala locations across Australia and the

549

implications for a recombinant C. pecorum major outer membrane protein based vaccine.

550

Veterinary Microbiology 167, 513-522.

551 552

Lee, K.E., Seddon, J.M., Johnston, S., FitzGibbon, S.I., Carrick, F., Melzer, A.,

553

Bercovitch, F., Ellis, W., 2013. Genetic diversity in natural and introduced island

554

populations of koalas in Queensland. Aust. J. Zool. 60, 303-310.

555 556

Lin, Y., Ritchea, S., Logar, A., Slight, S., Messmer, M., Rangel-Moreno, J., Guglani, L.,

557

Alcorn, J.F., Strawbridge, H., Park, S.M., Onishi, R., Nyugen, N., Walter, M.J., Pociask,

558

D., Randall, T.D., Gaffen, S.L., Iwakura, Y., Kolls, J.K., Khader, S.A., 2009. Interleukin-

559

17 is required for T helper 1 cell immunity and host resistance to the intracellular

560

pathogen Francisella tularensis. Immunity. 31, 799-810.

561 562

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using Real-

563

Time Quantitative PCR and the 2-∆ ∆CT method. Methods. 25, 402-408.

564

27

565

Longbottom, D., Coulter, L.J., 2003. Animal chlamydioses and zoonotic implications. J.

566

Comp. Pathol. 128, 217-244.

567 568

Loomis, W.P., Starnbach, M.N., 2002. T cell responses to Chlamydia trachomatis. Curr.

569

Opn. in Microbio. 5, 87-91.

570 571

Maher, I.E., Griffith, J.E., Lau, Q., Reeves, T., Higgins, D.P., 2014. Expression profiles

572

of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated

573

koala lymphocytes (Phascolarctos cinereus) by qRT-PCR. PeerJ.

574

http://dx.doi:10.7717/peerj.280.

575 576

Mascellino, M.T., Boccia, P., Oliva, A., 2011. Immunopathogenesis in Chlamydia

577

trachomatis infected women. ISRN Obst. Gynec. http://dx.doi:10.5402/2011/436936.

578 579

Mathew, M., Beagley, K.W., Timms, P., Polkinghorne, A., 2013a. Preliminary

580

characterisation of tumor necrosis factor alpha and interleukin-10 responses to Chlamydia

581

pecorum

582

http://dx.doi:10.1371/journal.pone.0059958.

infection

in

the

koala

(Phascolarctos

cinereus).

PLoS

ONE.

8,

583 584

Mathew, M., Pavasovic, A., Prentis, P.J., Beagley, K.W., Timms, P., Polkinghorne, A.,

585

2013b. Molecular characterisation and expression analysis of Interferon gamma in

586

response to natural Chlamydia infection in the koala, Phascolarctos cinereus. Gene 527,

587

570-577.

588

28

589

Miguel, R.D.V., Harvey, S.A., LaFramboise, W.A., Reighard, S.D., Matthews, D.B.,

590

Cherpes, T.L., 2013. Human female genital tract infection by the obligate intracellular

591

bacterium Chlamydia trachomatis elicits robust Type 2 immunity. PLoS ONE. 8,

592

http://dx.doi.10.1371/journal.pone.0058565.

593 594

Mohamad, K.Y., Rodolakis, A., 2010. Recent advances in the understanding of

595

Chlamydophila pecorum infections, sixteen years after it was named as the fourth species

596

of the Chlamydiaceae family. Vet. Res. 41, http://dx doi: 10.1051/vetres/2009075.

597 598

Morris, K., Prentis, P., O'Meally, D., Pavasovic, A., Taylor-Brown, A., Timms, P., Belov,

599

K., Polkinghorne, A., 2014. The koala immunological toolkit: sequence identification and

600

comparison of key markers of the koala (Phascolarctos cinereus) immune response. Aust.

601

J. Zool. (In Press).

602 603

O'Meara, C.P., Armitage, C.W., Harvie, M.C., Timms, P., Lycke, N.Y., Beagley, K.W.,

604

2013(a). Immunization with a MOMP-based vaccine protects mice against a pulmonary

605

Chlamydia challenge and identifies a disconnection between infection and pathology.

606

PLoS ONE. 8, http://dx.doi: 10.1371/journal.pone.0061962.

607 608

O'Meara, C.P., Armitage, C.W., Harvie, M.C., Andrew, D.W., Timms, P., Lycke, N.Y.,

609

Beagley, K.W., 2013(b). Immunity against a Chlamydia infection and disease may be

610

determined by a balance of IL-17 signaling. Immunol Cell Biol. 10, http://dx

611

.doi:1038/icb.2013.92.

612

29

613

Peters, A., Lee, Y., Kuchroo, V.K., 2011. The many faces of Th17 cells. Curr. Opin.

614

Immunol. 23, 702-706.

615 616

Polkinghorne, A., Hanger, J., Timms, P., 2013. Recent advances in understanding the

617

biology, epidemiology and control of chlamydial infections in koalas. Vet. Micro, 165,

618

214-223.

619 620

Savarin, C., Stohlman, S.A., Hinton, D.R., Ransohoff, R.M., Cua, D.J., Bergmann, C.C.,

621

2012. IFN-gamma protects from lethal IL-17 mediated viral encephalomyelitis

622

independent of neutrophils. J. Neuroinflammation. 9, http://dx.doi. 10.1186/1742-2094-9-

623

104.

624 625

Schneemilch, C.E., Schilling, T., Bank, U., 2004. Effects of general anaesthesia on

626

inflammation. Best. Pract. Res. Clin. Anaesthesiol. 18, 493-507.

627 628

Scurlock, A.M., Frazer, L.C., Andrews, C.W., Jr., O'Connell, C.M., Foote, I.P., Bailey,

629

S.L., Chandra-Kuntal, K., Kolls, J.K., Darville, T., 2011. Interleukin-17 contributes to

630

generation of Th1 immunity and neutrophil recruitment during Chlamydia muridarum

631

genital tract infection but is not required for macrophage influx or normal resolution of

632

infection. Infect. Immun. 79, 1349-1362.

633 634

Shemer-Avni, Y., Wallach, D., Sarov, I., 1988. Inhibition of Chlamydia trachomatis

635

growth by recombinant tumor necrosis factor. Infect. Immun. 56, 2503-2506.

636

30

637

Vats, V., Agrawal, T., Salhan, S., Mittal, A., 2007. Primary and secondary immune

638

response of mucosal and peripheral lymphocytes during Chlamydia trachomatis

639

infection. FEMS Immunol. Med. Microbiol. 49, 280-287.

640 641

Wan, C., Loader, J., Hanger, J., Beagley, K.W., Timms, P., Polkinghorne, A., 2011.

642

Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious

643

load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos

644

cinereus). Australian Veterinary Journal. 89, 409-412.

645 646

Wang, C., Tang, J., Geisler, W.M., Crowley-Nowick, P.A., Wilson, C.M., Kaslow, R.A.,

647

2005. Human leukocyte antigen and cytokine gene variants as predictors of recurrent

648

Chlamydia trachomatis infection in high-risk adolescents. J. Infect. Dis. 191, 1084-1092.

649

Yang, X., 2001. Distinct function of Th1 and Th2 type delayed type hypersensitivity:

650

protective and pathological reactions to chlamydial infection. Microsc. Res. Tech. 53,

651

273-277.

652 653

Ye, P., Garvey, P.B., Zhang, P., Nelson, S., Bagby, G., Summer, W.R., Schwarzenberger,

654

P., Shellito, J.E., Kolls, J.K., 2001. Interleukin-17 and lung host defense against

655

Klebsiella pneumoniae infection. Am. J. Respir. Cell Mol. Biol. 25, 335-340.

656 657

Yu, H., Jiang, X., Shen, C., Karunakaran, K.P., Jiang, J., Rosin, N.L., Brunham, R.C.,

658

2010. Chlamydia muridarum T-cell antigens formulated with the adjuvant DDA/TDB

659

induce immunity against infection that correlates with a high frequency of gamma

660

interferon (IFN-gamma)/tumor necrosis factor alpha and IFN-gamma/interleukin-17

661

double-positive CD4+ T cells. Infect. Immun. 78, 2272-2282.

662

31

663

Zhang, X., Gao, L., Lei, L., Zhong, Y., Dube, P., Berton, M.T., Arulanandam, B., Zhang,

664

J., Zhong, G., 2009. A MyD88-dependent early IL-17 production protects mice against

665

airway infection with the obligate intracellular pathogen Chlamydia muridarum. J.

666

Immunol. 183, 1291-1300.

667 668

Zhang, Y., Wang, H., Ren, J., Tang, X., Jing, Y., Xing, D., Zhao, G., Yao, Z., Yang, X.,

669

Bai, H., 2012. IL-17A synergizes with IFN-gamma to upregulate iNOS and NO

670

production and inhibit chlamydial growth. PLoS ONE. 7,

671

http://dx.doi: 10.1371/journal.pone.0039214.

672 673

Zhou, X., Chen, Q., Moore, J., Kolls, J.K., Halperin, S., Wang, J., 2009. Critical role of

674

the Interleukin-17/Interleukin-17 receptor axis in regulating host susceptibility to

675

respiratory infection with Chlamydia species. Infect. Immun. 77, 5059-5070.

676

32

677

Highlights

678

•

679 680

IL17A cytokine •

681 682 683

IL17A qrtPCR developed to provide the first functional analysis of a marsupial

Koalas with chlamydial disease had significantly higher IL17A gene expression compared to asymptomatically infected animals

•

Koalas with history of chlamydial disease had higher pro-inflammatory cytokine gene expression compared to healthy koalas

684

33