1
2 CXCL10, CXCL11, HLA-A, and IL-1 are induced in peripheral blood
3 mononuclear cells from women with Chlamydia trachomatis related infertility
4
5
6 Shruti Menon1, Kimberly Alexander1, Peter Timms2, John A Allan3,4, and Wilhelmina
7 M. Huston1,3
8
9 1 Institute of Health and Biomedical Innovation, Queensland University of
10 Technology, Brisbane, Australia
11 2Faculty of Science, Health, Education and Engineering, University of the Sunshine
12 Coast, Australia
13 3Wesley and St Andrews Research Institute, The Wesley Hospital, Auchenflower,
14 QLD, 4066, Australia.
15 4UC Health Clinical School, The Wesley Hospital, Auchenflower, QLD, 4066.
16
17
18
19
20
21
22
1 23 ABSTRACT
24 Chlamydia trachomatis (CT) infections can result in the development of serious
25 sequelae such as pelvic inflammatory disease and tubal infertility. In this study,
26 peripheral blood mononuclear cells (PBMC) from women who were undergoing or
27 had recently undergone IVF treatment were cultured ex vivo with C. trachomatis to
28 identify the immune responses associated with women who had serological evidence
29 of a history of Chlamydia infection. Cytokines secreted into the supernatant from the
30 cultures were measured using ELISA, and the level of IL-1 was found to be
31 significantly higher in Chlamydia positive women than Chlamydia negative women.
32 qRT-PCR analysis of expression of 88 immune related genes showed trends towards
33 an upregulation of CXCL10, CXCL11, and HLA-A in Chlamydia positive women
34 compared to Chlamydia negative women. These findings support that some women
35 launch a more marked pro-inflammatory response upon infection with C. trachomatis
36 and this may be associated with why C. trachomatis induces infertility in some
37 infected women.
38
39
40
41
42
43
44
45
46
47
2 48 INTRODUCTION
49 Chlamydia (C.) trachomatis is one of the most common sexually transmitted bacteria
50 in the world (reviewed (1)). The infection can result in serious complications in
51 women including, pelvic inflammatory disease (PID), ectopic pregnancy and tubal
52 infertility (2). C. trachomatis pathogenesis and the mechanism resulting in female
53 infertility have been investigated using various in vitro techniques; including infection
54 of HeLA cells with C. trachomatis where the expression of genes that regulate innate
55 immunity (3) and IL-8 (4) has been detected. Specific chlamydial antigens have also
56 been proposed to mediate much of the inflammation, in one example, Zhou et al. (5)
57 showed that expression of the C. trachomatis pORF5 protein in HeLa cells induced
58 the expression of TNF-, IL-1, and IL-8. Additionally ex vivo studies using infection
59 with C. trachomatis serovar D on primary cells isolated from human fallopian tube
60 showed that IFN- effector functions in hypoxic conditions facilitated the
61 development of chronic infections (6). Ex-vivo stimulation with C. trachomatis on
62 PBMC and endometrial tissue of women showed an accumulation of IL-4 (7) and a
63 polarization towards Type 2 immunity (8). Murine models have shown that infection
64 with C. trachomatis alters CD8+ T cell responses in eliciting protection against the
65 pathogen (9), and ex vivo studies on mouse macrophages and fibroblasts showed an
66 MyD88 dependent induction of IFN- and IP-10 (10). Additionally, when challenged
67 with C. trachomatis MoPn biovar in the ovarian bursa of C3H and C57BL/6 mice,
68 they developed hydrosalpinx that subsequently resulted in infertility (11). These
69 studies indicate that a pro-inflammatory response from the local tissue is likely a
70 major contributor to the pathological outcome, however the paradox is that a pro-
71 inflammatory or cellular response is essential to clear the intracellular chlamydial
72 infection.
3 73
74 Several studies have identified immune factors expressed from peripheral blood
75 mononuclear cells from Chlamydia infected or infertile participants. The stimulation
76 of PBMC from infertile women with chlamydia 60kDa heat shock protein (HSP60)
77 yielded higher production of IFN-, IL-10 and IL-12 cytokines (12). In vitro
78 lymphocyte proliferation of PBMC from women with salpingitis with C. trachomatis
79 57-kDa Hsp showed a production of cell-mediated responses to the infection,
80 indicating its possible role in tubal pathology (13). Pro-inflammatory or regulatory
81 cytokines such as IL-17(14), IFN- (15), IL-6 (16), and IL-22 (14) have been
82 previously shown to be secreted from C. trachomatis stimulated PBMC of patients
83 with acute infections, or chlamydial infertility.
84
85
86 Diagnosis of women with chlamydial infertility is typically conducted using surgical
87 or sonographic investigation for fallopian tubal blockage (17). However, even in the
88 absence of apparent tubal occlusion women who are sero-positive for CT are 50% less
89 likely to conceive other than by IVF treatment (18). Approximately 45% of tubal
90 infertility is due to C. trachomatis (19). However, in spite of the prevalence of this
91 condition we still only have limited understanding of the disease mechanism that
92 results in infertility in some women.
93
94 Here PBMC from infertile female participants who are undergoing or had recently
95 undergone IVF treatment were isolated and cultured ex vivo in the presence of C.
96 trachomatis. The gene expression of 88 innate and adaptive genes and 10 secreted
97 cytokines were measured from the cultures to detect differences in the responses from
4 98 women who had serological evidence of chlamydial infertility compared to those with
99 other causes of infertility.
100
101 MATERIALS AND METHODS
102 Whole blood and sera was collected from fully consented voluntary participants. 31
103 women with infertility (greater than 1 year of attempting to conceive and requiring
104 IVF treatment) who were attending an IVF clinic or who were pregnant having
105 recently conceived from treatment at the IVF clinic participated in the study. The
106 study was reviewed and approved by the Queensland University of Technology and
107 UC Health Human Research Ethics Committees: UC Health Ethics approval 1314,
108 QUT Human Research Ethics approval 1300000505. Chlamydia
109 Microimmunofluorescence (MIF) IgG (Focus Diagnostics, USA) assay was
110 conducted on the participant sera to determine the C. trachomatis infection history
111 status.
112
113 C. trachomatis D (ATCC VR-885) and F strain (ATCC VR-346) were cultured in
114 McCoy cells. Confluent cells were infected with the strains and incubated at 37C for
115 44 hours. Following infection, the strains were semi-purified. To prepare a mixture of
116 C. trachomatis D and F strains, the cultures were mixed together (equal ratio of EBs)
117 and purified using density gradient centrifugation (29% v/v urografin Ultravist®
118 (Sigma-Aldrich)), and stored in sucrose phosphate buffer at -80C.
119
5 120 Peripheral blood mononuclear cells were isolated from participant whole blood.
121 PBMC were isolated using Ficoll-PlaqueTM Premium reagent (GE Healthcare) as
122 previously described in Cunningham et al. (16). PBMC (1 x 106cells/well) and they
123 were stimulated at an MOI of 5 with purified C. trachomatis serovar D (ATCC: VR-
124 885), purified C. trachomatis serovar F (ATCC VR-346), purified C. trachomatis D
125 and F mix (50:50 elementary bodies (EBs)), phorbol myristate acetate (PMA)
126 (positive control) and media (negative control). The cells were incubated for 15 hours
127 at 37C. After incubation, the cells were centrifuged at 800 × g, and the supernatant
128 was collected. The cells were resuspended in RNA cell protect® (Qiagen, Victoria,
129 Australia) for gene expression analysis. The levels of IFN-, IL-12, IL-1B, IL-2, IL-4,
130 IL-6, IL-8, IL-10, IL-17 and TNF- were measured from the supernatants using
131 ELISA kits (Elisakit.com, Melbourne, Australia) as per manufacturer’s instructions.
132 Total RNA was extracted and purified using Qiagen RNeasy Micro Kit (Victoria,
133 Australia), and reverse transcribed into cDNA using Qiagen RT2 First strand kit and
134 expression of innate and adaptive immune genes were determined using Qiagen RT2
135 Profiler PCR arrays. The expression levels of all genes were normalized to 5
136 housekeeping genes (ACTB, B2M, GAPDH, RPLP0 and HPRT1) for each participant
137 sample as per manufacturer’s instructions. The fold change between CT positive
138 infertile women and CT negative infertile women were determined using 2-Ct
139 method(20).
140
141 RESULTS
142 Participants were classified as serologically positive for C. trachomatis using
143 Chlamydia trachomatis MIF IgG assay (four out of 31 were MIF positive and
144 considered Chlamydia infertile). All participants were women undergoing or recently
6 145 had undergone IVF treatment. The participants who were sero-negative to C.
146 trachomatis infection were characterized as CT negative (n=27). The immune
147 responses were then compared to media as a control C. trachomatis D and F strain
148 treatments in the same groups using cytokine ELISAs and qRT-PCR analysis. Figure
149 1 shows the level of secreted cytokines from the supernatants of both Chlamydia
150 positive and Chlamydia negative infertile women after culture in the presence of C.
151 trachomatis D and F strains. In response to the culture in the presence of the C.
152 trachomatis D and F EBs, IL-8 (617.5pg/mL versus 602.5pg/mL) and TNF- (111
153 pg/mL versus 71.73pg/mL) were produced in both cohorts; however, only IL-1
154 (96.42pg/mL versus 41.42pg/mL) production in Chlamydia positive women was
155 significantly higher than Chlamydia negative women (p<0.05). Compared to the
156 negative control (unstimulated, media only control) for Chlamydia positive infertile
157 women, the levels of IL-8 (617.5pg/mL and 515pg/mL), TNF- (111pg/mL and
158 7.557pg/mL) and IL-1 (96.42 pg/mL vs 7.76pg/mL) were significantly higher in the
159 PBMC stimulated with C. trachomatis D and F strains (Figure 2). Therefore, this is a
160 reflection of the Chlamydia response of the PBMCs from these participants rather
161 than a general response of the PBMCs to culture.
162
163 Expression of a selection of 88 innate and adaptive immune genes from PBMC
164 cultured with C. trachomatis D and F was analysed using a RT-PCR array. The gene
165 expression was normalized to housekeeping genes for each participants’ culture and
166 the fold change in gene expression between C. trachomatis infertile women and
167 women with infertility for other reasons tested (ct). Three genes showed a trend
168 towards higher expression levels in CT positive infertile women (n=4) compared to
169 CT negative infertile women (n=27) (Table 2). The chemokines CXCL10 (5.48-fold)
7 170 and CXCL11 (2.31-fold), and human leukocyte antigen HLA-A (2.22-fold) showed
171 the most notable differences between the two participant groups (although these were
172 not significantly different, possibly due to the low sample size of CT positive infertile
173 women).
174
175
176
177 DISCUSSION
178
179 The cytokines secreted and immune gene expression in peripheral blood mononuclear
180 cells was tested to provide information into the immune pathways that are activated
181 from women who have C. trachomatis infertility. Within 15 hours of co-culture with
182 C. trachomatis, three immune genes were expressed at greater than 2 fold higher
183 levels in Chlamydia positive infertile women compared to Chlamydia negative
184 infertile women (not significant). A mix of two well characterized and common C.
185 trachomatis strains were used in the co-culture model, rather than a single strain,
186 although these may not have been the same as the strain that originally infected the
187 participants to cause the infertility. These two strains will not cover all of the relevant
188 diversity of C. trachomatis, but were used to provide more diversity in the simulating
189 antigen mix than a single strain. Infection by C. trachomatis is likely to trigger a
190 range of immune responses as a result of antigen binding human leukocyte antigens
191 (HLA) molecules. Previous studies have elucidated the role of HLA class I and class
192 II molecules and their genotypes in increasing the susceptibility to tubal pathology
193 and infertility caused by C. trachomatis in women (21-25). In this study, the HLA-
194 class I molecule, HLA-A gene was upregulated in CT positive infertile women (not
8 195 significant). Kimani et al., (21) reported that HLA-A31 allele was a significant risk
196 factor for C. trachomatis PID in a longitudinal study of urban female sex worker in
197 Kenya (n=23 PID cases). Additionally, HLA-A2 was reported to elicit cytotoxic T
198 lymphocyte responses to C. trachomatis MOMP in peripheral blood of Chlamydia
199 infected patients (26).
200
201 Our study showed an increased expression of two chemokines CXCL10 (IP-10) and
202 CXCL11 in women with Chlamydia infertility compared to the women with infertility
203 for other reasons. Chemokines are small pro-inflammatory molecules that induce
204 migration of leucocytes (27). The role of CXCL10 in protective immunity against
205 chlamydial infections has been implicated in murine model infected with C.
206 muridarum (28). In polarized polA2EN epithelial cells, a reduction of CXCL10 was
207 observed on infection with C. trachomatis (29); whereas in A2497 epithelial cells, an
208 upregulation of CXCL10 was observed (30). CXCL10 recruits CXCR3 and CCR5
209 positive leukocytes such as T cells and natural killer cells to the site of infection (29).
210 Previous studies on mouse models have shown that these chemokines are
211 predominantly expressed in upper genital tract infection and mediate protection
212 through Th1 or pro-inflammatory immunity (28, 31, 32). Wan et al. (33) showed that
213 CXCL11 (IFN-inducible T cell -chemoattractant or i-TAC) gene expression,
214 stimulated by the interferons (34), were higher in secretory phase cells on infection
215 with C. trachomatis.
216
217 Whilst a trend towards an increase in mRNA levels were observed for IL-1 and
218 TNF- (not significant), ELISAs detection of the secreted protein showed that
219 secreted IL-1 was significantly higher in Chlamydia positive infertile women than
9 220 Chlamydia negative infertile women (p<0.05). Supporting the previous findings of
221 Hvid et al. (35), this study also shows that IL-1 is an important proinflammatory
222 cytokine induced by C. trachomatis infection, and therefore likely an important
223 contributor to pathology from C. trachomatis infection. Here the IL-1 was detected
224 from primary ex vivo culture of PBMC rather than reproductive tract epithelia (as in
225 Hvid et al., (35)), suggesting that both the innate epithelial response and innate
226 mononuclear response from some women in response to C. trachomatis infection is
227 pro-inflammatory and involves IL-1. Both IL-1 and TNF- are potent inducers of
228 IL-8, hence high levels of the cytokine were observed in the supernatants of
229 Chlamydia positive and Chlamydia infertile women (36).
230
231 The limitations of our study include a small sample size that might account for the
232 lack of significance between the cohorts for many factors. The observed differences in
233 responses here for women with Chlamydia infertility compared to women with other
234 causes of infertility supports the model that some women launch a more marked pro-
235 inflammatory response to the infection and thus develop the serious pathology and
236 disease sequelae and these responses may reflect the original response to the infection
237 in the local tissue.
238
239 FUNDING
240 This work was partially funded by a Queensland Government Smart State NIRAP
241 project.
242
243 ACKNOWLEDGEMENTS
244
10 245 The authors would like to thank the voluntary participants involved in this study. The
246 authors acknowledge the involvement of Ms Helen Woodhouse in participant
247 recruitment. The Wesley Research Institute Tissue Bank was involved in sample
248 collection.
249
250 CONFLICT OF INTEREST
251 The authors declare that there are no conflicts of interest.
252
253
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375
376 377 378 Figure 1: The graph shows the concentration of cytokines detected in the
379 supernatants of the ex vivo PBMC cultures in the presence of C. trachomatis D
380 and F. Chlamydia positive infertile women (n=4) and Chlamydia negative infertile
381 women (n=27) (* indicates p<0.05).
382 383 Figure 2: The graph shows the concentration of cytokines detected in the
384 supernatants of the ex vivo PBMC cultures of Chlamydia positive infertile women
385 in the presence of C. trachomatis D and F and control cultures (no Chlamydia
386 added) (* indicates p<0.05).
387 388 389 390
16 391 392 Table 1: Participant fertility data
CT Mean Type of infertility IVF outcome Gravidas past Gravidas at
serology Age *ectopic (n=) *success(n=) *nulliparous(n=) the time of
status by (years) *tubal(n=) *failure (n=) *multiparous (n=) collection
MIF (n=)
CT 39.5 *Tubal (n=3); PID Success (n=2) Nulliparous (n=3) n=0
positive (n=1); salpingitis Failure (n=2) Multiparous (n=1)
infertile (n=1); tubal adhesion
women (n=2)]
(n=4) *Unknown etiology
(n=1)
CT 37.4 *Tubal (n=4); ovary Success (n=11) Nulliparous (n=13) n=1
negative removal (n=2); PID Failure (n=8) Multiparous (n=6)
infertile (n=1); salpingitis
women (n=1); tubal
(n=19) obstruction (n=1);
ovarian cystecty
(n=1); Sterile (n=1);
*Ectopic pregnancy
(n=1)
*Polycystic ovarian
syndrome (PCOS)
(n=4)
17 *Endometritis (n=4)
+ [PID=(n=2)]
*Unknown etiology
(n=6);
393 394
18 395
396 Table 2: Gene expression of various immune related genes from PBMC cultures
397 with C. trachomatis from Chlamydia positive infertile women
398
Gene Gene function (37) Fold change (CT positive infertile vs CT negative infertile)* (2-CT) CXCL10 CXC motif ligand 10 5.480 CXCL11 chemotactic for T cells 2.319 HLA-A Human leucocyte antigen 2.224 MX1 guanosine triphosphate metabolizing protein 2.044 TNF Tumour necrosis factor. 1.689 HLA-E Human leucocyte antigen 1.317 C3 Complement component 3 1.190 NFKBIA Nuclear factor of Kappa light polypeptide gene 1.105 IRF3 Interferon regulatory transcription factor 0.185 IL1B Interleukin 1 cytokine 1.08 CCL5 Chemokine, chemoattractant 1.055 STAT3 Transcription factor 1.010 IL8 Chemoattractant, 0.995 FASLG tumor necrosis factor superfamily FAS/FASLG 0.988 signaling pathway DDX58 DEAD box proteins, putative RNA helicases 0.962 STAT1 Signal transducer and activator of transcription 0.926 MYD88 Cytosolic adapter protein 0.913 IFNAR1 Receptor for interferons alpha and beta 0.912 CD14 Surface antigen 0.875 IL1A Interleukin 1 cytokine family 0.854 STAT4 Signal transducer and activator of transcription 0.831 CSF2 Colony stimulating factor 2 0.818 IL23A Subunit of cytokine interleukin 23 0.782 NFKB1 Transcription regulator 0.744 ICAM1 Intercellular adhesion molecule 1 cell surface 0.710 glycoprotein CD40 TNF-receptor superfamily; receptor on antigen- 0.689 presenting cells CXCR3 G protein-coupled receptor 0.629 IL6 Interleukin 6 0.622 TBX21 transcription factor 0.591 CASP1 caspase 0.580
19 CD80 Membrane receptor 0.578 IRF7 interferon regulatory factor 7 0.546 STAT6 Signal transduction and transcription 0.532 IFNG Type II interferon family 0.519 IFNGR1 gamma interferon receptor 0.515 MAPK1 MAP kinases 0.448 ITGAM integrin alpha M chain 0.441 IFNA1 Interferon alpha 0.4068 CD8A cell surface glycoprotein 0.403 IFNB1 Interferon beta 0.394 TRAF6 TNF receptor associated factor 0.392 NOD1 nucleotide-binding oligomerization domain 0.374 CXCR5 CXC chemokine receptor family 0.359 CD4 Membrane glycoprotein 0.340 CD86 cell surface ligand 0.323 MAPK8 MAP kinases 0.310 CCL2 Chemokine 0.294 IL10 Cytokine 0.292 TICAM1 Adaptor protein containing (TIR) 0.271 JAK2 Janus kinase 2 0.257 LYZ Lysozyme 0.257 TLR3 Toll-like receptor 0.2541 IL2 Interleukin 2 0.225 CCR6 Beta chemokine receptor 0.211 TLR7 Toll-like receptor 0.204 IRAK1 Interleukin-1 receptor-associated kinase 0.198 CD40LG Surface of T cells 0.191 SLC11A1 Solute carrier family 11 0.183 FOXP3 transcriptional regulator 0.162 NLRP3 Upstream activator of NF-kappaB 0.159 TLR2 Toll-like receptor 0.143 CCR5 Beta chemokine receptor family, 0.142 IL18 Proinflammatory cytokine 0.138 IL1R1 Cytokine receptor interleukin-1 alpha, 0.117 TLR1 Toll-like receptor 0.098 TLR4 Toll-like receptor 0.098 LY96 associates with toll-like receptor 4 0.089 NOD2 Nod1/Apaf-1 family 0.088 TLR8 Toll-like receptor 0.086 TLR6 Toll-like receptor 0.081 RAG1 Involved in activation of immunoglobulin V-D- 0.081 J recombination IL5 Interleukin 5 cytokine 0.080 GATA3 transcription factors 0.080
20 IL17A Proinflammatory cytokine 0.078 TLR5 Receptor mobilizes the nuclear factor NF- 0.076 kappaB CXCL13 Chemokine 0.075 IL4 Interleukin 4 0.073 MBL2 Soluble mannose-binding lectin 0.073 APCS Glycoprotein 0.072 TLR9 Toll-like receptor 0.072 CRP Host defence related functions 0.071 IL22 Interleukin-22 0.045 IL13 immunoregulatory cytokine; B-cell maturation 0.041 MPO Myeloperoxidase 0.037 CCR4 G-protein-coupled receptor family; receptor for 0.036 the CC chemokine TYK2 tyrosine kinase Janus kinases (JAKs) 0.034 CCR8 beta chemokine receptor family 0.015 RORC DNA-binding transcription factor 0.081
399 *CT was obtained by normalizing the level of expression of gene of interest to the GOI AVG HKG 400 expression level of housekeeping genes (HKG) (CT= CT - CT ). Fold change 401 in gene expression (2-CT) was determined by dividing the normalized expression of 402 gene of interest of the experimental sample by the normalized expression of the same
403 gene of interest in the control sample; where CT= CT (CT positive infertile
404 sample)- CT (CT negative infertile sample). 405
406 407
21 408 409 Fig 1 410 411 412 413 414
415 416 Fig 2
22