Chlamydia suis and Chlamydia trachomatis induce multifunctional CD4 T cells in pigs

Vaccine 35 (2017) 91–100

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Chlamydia suis and Chlamydia trachomatis induce multifunctional CD4 T cells in pigs T. Käser a,b,⇑, J.A. Pasternak a, M. Delgado-Ortega a, G. Hamonic a, K. Lai a, J. Erickson a, S. Walker a, J.R. Dillon a, V. Gerdts a, F. Meurens c,d a Vaccine and Infectious Disease Organization – International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, 120 Veterinary Road, S7N 5E3 Saskatoon, Saskatchewan, Canada b Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, 27607 Raleigh, NC, USA c LUNAM Université, Oniris, Nantes-Atlantic College of Veterinary Medicine and Food Sciences and Engineering, UMR BioEpAR, F-44307 Nantes, France d INRA, UMR1300 Biology, Epidemiology and Risk Analysis in Animal Health, CS 40706, F-44307 Nantes, France

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Article history: Received 24 August 2016 Received in revised form 7 November 2016 Accepted 10 November 2016 Available online 25 November 2016 Keywords: Chlamydia suis Chlamydia trachomatis Animal model Swine Vaccine development One Health

a b s t r a c t Chlamydia trachomatis infections are the most prominent bacterial sexually-transmitted disease worldwide and a lot of effort is put into the development of an effective vaccine. Pigs have been shown to be a valuable animal model for C. trachomatis vaccine development. The aim of this study was to decipher the T-cell-mediated immune response to chlamydial infections including C. trachomatis and C. suis, the chlamydia species naturally infecting pigs with a demonstrated zoonotic potential. Vaginal infection of pigs with C. suis and C. trachomatis lasted from 3 to 21 days and intra-uterine infection was still present after 21 days in 3 out of 5 C. suis- and 4 out of 5 C. trachomatis-inoculated animals and caused severe pathological changes. Humoral immune responses including neutralizing antibodies were found predominantly in response to C. suis starting at 14 days post inoculation. The T-cell-mediated immune responses to C. trachomatis and C. suis-infections started at 7 days post inoculation and consisted mainly of CD4+ T cells which were either IFN-c single cytokine-producing or IFN-c/TNF-a double cytokine-producing Thelper 1 cells. IL-17-producing CD4+ T cells were rare or completely absent. The T-cell-mediated immune responses were triggered by both homologous or heterologous re-stimulation indicating that crossprotection between the two chlamydia species is possible. Thus, having access to a working genital C. suis and C. trachomatis infection model, efficient monitoring of the host-pathogen interactions, and being able to accurately assess the responses to infection makes the pig an excellent animal model for vaccine development which also could bridge the gap to the clinical phase for C. trachomatis vaccine research. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Besides in silico and in vitro studies, vaccine development mostly relies on animal models and choosing the right animal model is crucial for a successful translation into humans. Mice are the most frequently used animal model but before going into clinical trials, vaccine candidates should be tested in at least one other species. Large animal models are particularly beneficial for predicting the vaccine outcome in humans [1]. Thus, many studies use non-human primate (NHP) models to test promising vaccine candidates but although NHPs deliver valuable information the use of NHPs is very controversial due to their high economical ⇑ Corresponding author at: Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, 27607 Raleigh, NC, USA. E-mail address: [email protected] (T. Käser). http://dx.doi.org/10.1016/j.vaccine.2016.11.050 0264-410X/Ó 2016 Elsevier Ltd. All rights reserved.

and ethical costs [1,2]. Pigs have several advantages as large animal models for vaccine design and testing including a similar physiology and immune system [1,3]. The porcine female genital tract shows several similarities to humans in regard to the reproductive cycle and immune system [4] making pigs a valuable animal model for studying sexually transmitted infections (STIs) like Chlamydia trachomatis (Ct) infections. Ct infections are the most prevalent bacterial STIs worldwide, and cause long-term complications such as infertility, chronic-pelvic pain and ectopic pregnancies [5]. So far, Ct infections are screened and treated by antibiotics with limited success and a lot of effort is put in the development of a protective vaccine. To date, this effort includes three studies on Ct vaccine candidates in pigs [6–8] and two more recent studies in mini-pigs [9,10] showing promising results by a decreased bacterial load and the induction of IFN-c, mucosal IgA and serum neutralization

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antibodies. But due to previous limitations in the porcine immunological toolbox, the immune response analysis of antigen-specific CD4+ TH1 cells was limited to investigating IFN-c production in ELISAs without determining its cell source or examining bulk CD4+ T cells in blood of infected animals without identifying chlamydia-specific cells. Antigen-specific CD4+ TH1 cells, however, are crucial for the control and elimination of infection, and for the induction of a protective memory immune response in Ct infections [11,12]. Thus, the aim of this study was to analyse chlamydia infection and the resulting immune response in pigs with a focus on the CD4+ T-cell immune response including the detection of multifunctional CD4+ T cells. These cells possess a robust effector function, have a high long-term memory potential and have been shown to be good correlates of protection in C. muridarum infections [13]. We also included C. suis (Cs) in our studies, due to its high prevalence in pigs, similar disease outcomes as Ct, and the zoonotic potential demonstrated by its presence in pharyngeal and rectal samples of slaughterhouse employees [14]. 2. Materials and methods

2.4. Preparation of single cell suspensions from lymph nodes Lymph nodes were cut into small pieces and pressed through cell strainers (BD Biosciences) into 50 ml tubes. Cell strainers were flushed with ice-cold PBS to elute the single cell suspensions. 2.5. Genital tract handling Genital tracts were examined for gross pathological changes. Then, uterine horns were flushed with 40 ml PBS for immunotyping of infiltrated immune cells via FCM and for the detection of chlamydia in the uterine horns via qPCR. Horn flush cells were pelleted via centrifugation and 1 ml of the supernatant was used for chlamydia detection via qPCR upon treatment as described for the swab samples above. The pelleted cells were immunotyped via FCM as described below. Biopsy samples of altered tissue were homogenated using a Mini Bead Beater with zirconia beads (BioSpec Products, OK) and lysed in an alkaline lysis buffer. DNA content was determined using a NanoDrop spectrophotometer (ThermoFisher Scientific) and 200 ng DNA were subsequently used per qPCR reaction.

A detailed description of the materials and methods can be found in Supplementary Information 1.

2.6. Detection of chlamydia via qPCR

2.1. Chlamydiaceae

Chlamydial DNA content was analysed via Taqman qPCR assay as described before [18]. A standard curve of Cs EBs was included on every plate in order to determine the concentration of chlamydial EBs in the test samples.

The Cs strain S45, originally isolated in Austria [15], and Ct serovar E strain Bour, a Ct reference strain with high prevalence [16], were purchased from ATCC and grown on HeLa cells using standard techniques [17] and purified as previously described [18]. Titration was performed on HeLa cells and infection was analysed via flow cytometry (FCM) as previously described [19]. 2.2. In vivo infection experiment Fifteen synchronized 26-week-old female pigs were inoculated in standing estrous intra-vaginally and intra-uterine each with 108 inclusion forming units (IFU) Cs or Ct in sucrose-phosphateglutamine (SPG) buffer, or MOCK-inoculated with SPG buffer alone. Clinical monitoring and rectal temperature recording was performed every other day. Blood, serum and vaginal swabs were collected at 3, 7, 10 and 14 dpi and at necropsy which was performed on three consecutive days 21, 22 and 23 dpi, collectively referred to as ‘‘21 dpi” (Fig. 1). Pigs were sacrificed using a captive bolt gun followed by exsanguination. At necropsy, the genital tracts and draining lymph nodes (iliac and uterine) were collected to monitor gross pathological changes and for sample collection. Experimental procedures are in accordance with the Animal Research Ethics Board (AREB) of the University of Saskatchewan (AREB # 20130095).

2.7. Immunotyping in blood, draining lymph nodes and uterine horn flushes Single-cell suspensions were stained for T-cell subset discrimination markers as summarized in Table 1. Cells were recorded on a FACS Calibur using the CELLQuest Software (BD Biosciences) and data analysis was performed with FlowJo version 7.6.5 (FLOWJO LLC, Ashland, OR). 2.8. Serum IgG detection Anti-chlamydial MOMP serum IgG levels were detected via ELISA. A mix of three partial MOMP proteins (ab67705-ab67707, Abcam, Cambridge, MA) were used for plate coating, before a 1:100 dilution of serum in PBS was added. Anti-pigIgG(H+L)alkaline phosphatase (KPL, Gaithersburg, MD) in combination with p-nitrophenyl phosphate (PNPP, ThermoFisher Scientific) was used for the color substrate reaction and color change was recorded on an iMark microplate reader (BioRad, Hercules, CA). An internal control was included on every plate and data were normalized to this control in order to compensate for plate-to-plate variations.

2.3. Swab, serum and blood sampling

2.9. Neutralizing antibody determination

Swab samples were collected into 1 ml of PBS, shaken for 1 h at 4 °C and spun for 1 h at 13,000g to pellet chlamydial elementary bodies (EBs). The pellet was lysed in 100 ll alkaline lysis buffer, neutralized and 2 ll of the supernatant was used for subsequent qPCR analysis. For serum collection, blood was collected into SST tubes (BD Biosciences, San Jose, CA), incubated for at least 30 min, spun for 20 min at 2000g and 20 °C and stored at
20 °C. Complete blood counts (CBCs) were determined from EDTA blood using a Hemavet 950 system (Drew Scientific Group, Miami Lakes, FL). PBMC isolation was performed via density gradient centrifugation.

Neutralizing antibodies in serum were detected by incubating heat-inactived serum with Cs elementary bodies (EBs) for 30 min at 37 °C in a final serum dilution of 1:10. This mixture was used to infect HeLa cells and infection was analysed via flow cytometry as described elsewhere [19]. Percent suppression was calculated for each animal using the following formula:

 % suppression ¼ 100


 % infection ½x dpi  100 % infection ½
2 dpi

where ‘‘x dpi” is the day of the calculated percent suppression.

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Infection: - MOCK - C. suis - C. trachomatis

Animal arrival Synchronization dpi: -20

-6 -2

0

3

7

Necropsy

10

14

21-23 Tissue: - Gross pathology - Chlamydia detection - Leukocyte infiltration - Lymphocyte isolation*

Clinical monitoring incl. temperature and vaginal discharge

Vaginal swab collection for the detection of chlamydia

Blood collection: - Total leukocyte counts - PBMC isolation* - Serum IgG levels - Neutralizing antibodies

* PBMC and lymph node cells were used for ex vivo phenotyping and in vitro restimulation Fig. 1. Setup of the in vivo experiment. Sexually mature pigs were synchronized and inoculated intra-vaginal and intra-uterine in standing estrous with MOCK, Cs and Ct (each 108 IFUs) at 0 days post inoculation (dpi). Pigs were clinically monitored every other day throughout the study and swab and blood collection was performed on the stated dpi. Necropsy was performed on 21–23 dpi (in the manuscript referred as 21 dpi) with additional gross-pathological analysis and sample collection from genital tissue and the draining uterine and iliac lymph nodes. Chlamydia were detected in swab samples, uterine horn flushes and tissue from pathologically changed tissue via qPCR. Immunotyping was performed in blood, lymph nodes and uterine horn flushes. IgG levels and neutralizing antibodies were determined in serum. The analysis of the T-cellmediated immune response analysis was performed in PBMC and cells from the draining lymph nodes.

Table 1 FCM antibody panels. Antigen

a b

Labeling strategy

Primary Ab source

2nd Ab source

Immunotyping in blood, draining lymph nodes and uterine horn flushes (T-cell subsets)a CD3 BB23-8E6-8C8 IgG2a PE CD4 74-12-4 IgG2b Alexa 488 CD8a 76-2-11 IgG2a Alexa 647 TCR-cd PGBL22A IgG1 PerCP-Cy5.5

Clone

Isotype

Fluorochrome

Directly conjugated Secondary antibody Secondary antibody Biotin-streptavidinb

BD Biosciences Kingfisher Biotech. Kingfisher Biotech. Kingfisher Biotech.

– Southern Biotech Southern Biotech Southern Biotech

Immunotyping in blood, draining lymph nodes and uterine horn flushes (Tregs) CD3 BB23-8E6-8C8 IgG2a FITC CD4 74-12-4 IgG2b Alexa 647 Foxp3 FJK-16s Rat IgG2a PE

Directly conjugated Secondary antibody Directly conjugated

BD Biosciences Kingfisher Biotech. eBioscience

– Southern Biotech –

Proliferation analysis of T-cell subsets CD4 74-12-4 CD8b PG164A TCR-cd PGBL22A

IgG2b IgG2a IgG1

PerCP-Cy5.5 Alexa 647 PE

Biotin-streptavidinb Secondary antibody Secondary antibody

BD Biosciences Kingfisher Biotech. Kingfisher Biotech.

Southern Biotech Southern Biotech Southern Biotech

Cytokine production of T-cell subsetsa CD3 BB23-8E6-8C8 CD4 74-12-4 CD8a 76-2-11 TCR-cd PGBL22A IFN-c P2G10 TNF-a MAb11 IL-17 SCPL1362

IgG2a IgG2b IgG2a IgG1 IgG1 IgG1 IgG1

PE-Cy7 PerCP-Cy5.5 Alexa 488 BrilliantViolet 421 PE BrilliantViolet 605 Alexa 647

Directly conjugated Biotin-streptavidinb Secondary antibody Secondary antibody Directly conjugated Directly conjugated Directly conjugated

BD Biosciences BD Biosciences Kingfisher Biotech. Kingfisher Biotech. BD Biosciences Biolegend BD Biosciences

– Southern Biotech Southern Biotech Biolegend – – –

Free binding sites of secondary antibodies were blocked by mouse IgG (Jackson Immuno Research, West Grove, PA) prior to direct antibody staining. Streptavidin-PerCP-Cy5.5 was purchased from BD Biosciences.

2.10. Antigen-specific T-cell subset proliferation Single cell suspensions were stained with CellTraceTM Violet Cell Proliferation Kit (ThermoFisher Scientific) and cultured for four days in the absence (medium) or presence of 100 ng/ml Cs or Ct lysate. After cultivation, the replicates were pooled and stained for cell-surface T-cell-subset differentiation antigens as summarized in Table 1. A total of 100,000 PBMC were recorded on a FACS Calibur using the CELLQuest Software (BD Biosciences). Data analysis was performed with FlowJo version 7.6.5 (FLOWJO LLC). 2.11. Antigen-specific cytokine production by T-cell subsets PBMC were cultured in vitro for 24 h in the absence (medium) or presence of 200 ng/ml Cs or Ct lysate, and Monensin (5 lg/ml,

Biodor Holding AG, Laufelfingen, CH) was added for the last 6 h of cultivation. Cultured cells were stained for T-cell markers and cytokines, and with the Near IR LIVE/DEADÒ Fixable Dead Cell Stain Kit (ThermoFisher Scientific) using the reagents described in Table 1. On average, >300,000 living single cells were recorded on a CyAnTM ADP Analyzer flow cytometer using the Summit Software (Beckman Coulter, Brea, CA). Data analysis was performed with FlowJo version 7.6.5 (FLOWJO LLC). 2.12. Statistical analysis Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). Data were analysed for normal distribution using the Kolmogorov-Smirnov test showing that data in most groups were normally distributed. Data recorded

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throughout the study were analysed using a repeated-measures two-way ANOVA with time and in vivo infection as the two factors. Data recorded only at necropsy were analysed by a standard twoway ANOVA with in vitro re-stimulation and in vivo infection as the two factors. Post hoc multiple comparisons were performed using the Dunnett’s test. Non-normally distributed data were analysed using a Mann-Whitney test. Differences were defined significant (⁄) for P < 0.05.

the uterine horns at 21 dpi. One Cs-inoculated pig had a chlamydia-concentration below the standard curve and two pigs were negative for Cs in the uterine horns at necropsy. In the Ctinoculated group, 4 out of 5 animals had low chlamydia levels in the uterine horns and 1 out of 5 animals was negative for Ct at necropsy.

3. Results

Results of the clinical monitoring and pathological analysis are summarized in Supplementary Fig. 1. With one exception, pigs did not show fever, clinical scores or inflamed vulvas. Early vaginal discharge (3 dpi) is normal at this stage of the reproductive cycle but one to two out of five Cs-inoculated animals had prolonged vaginal discharged. At necropsy, MOCK-inoculated pigs did not show abnormal histotroph, mucus production, or pathological changes with the exception of a congestion in one animal. Abnormal vaginal mucus was limited to one Ct-inoculated animal but cervical mucus was present in 3 out of 5 and 2 out of 5 Cs- and Ct-inoculated pigs, respectively (Supplementary Fig. 1A). While uterine horn flushes from MOCK-inoculated pigs were clear (data not shown), uterine horn flushes of 3/5 and 4/5 Cs- and Ct-inoculated pigs were cloudy indicating an abnormal histotroph with the presence of cells and debris in the upper genital tracts of these pigs (Supplementary Fig. 1A and B). Besides abnormal histotroph and mucus production, Cs- and Ct-inoculated pigs also showed considerable pathological changes like congestion throughout the genital tract, and low muscle tone or large vacuoles in the uterine horns (Supplementary Fig. 1A and C). Chlamydial DNA could be detected by qPCR in tissue samples of pathological changes and mucus from Cs- and Ct-inoculated animals indicating that the chlamydia infection was responsible for the pathological changes (data not shown).

3.1. Vaginal and uterine chlamydia infection Pigs were inoculated intra-vaginally and intra-uterine with MOCK, Cs or Ct. Intra-vaginal infection was monitored via qPCR of vaginal swabs (Fig. 2A). With two exceptions below or at the lowest detected concentration in the standard curve, all MOCKinoculated animals were negative throughout the study. Before inoculation (
2 dpi), Cs- and Ct-inoculated animals were negative. At 3 dpi, all Cs- and Ct-inoculated pigs were positive. At 7 dpi, two Cs inoculated pigs maintained their infection levels, one had a decreased Cs concentration and two pigs cleared the vaginal Cs-infection. Both pigs with high vaginal Cs load at 7 dpi stayed positive also at 14 dpi. At 21 dpi, one of these animals had cleared the infection while the Cs load in the other animal had even increased. In the Ct-inoculated group, 3/5 inoculated pigs had low vaginal Ct levels at 7 dpi while infection was cleared in the other two pigs. Intra-uterine infection was determined at necropsy (21 dpi) by qPCR analysis of uterine horn flushes (Fig. 2B). MOCK-inoculated pigs were negative except one pig with a chlamydia concentration below the standard curve. The two Cs-inoculated pigs which had vaginal Cs infections post 7 dpi were also highly positive for Cs in

3.2. Clinics and pathology

Chlamydia detection in swabs

Chlamydia in horn flushes

10

2

10

1

10

0

Chlamydia / horn

3

-2

dp i

10

21

4

dp i

10

14

5

dp i

10

7

6

dp i

10

3

10

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dp i

Chlamydia / ml swab

A) 10 8

10

8

10

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6

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10

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10

3

10

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10

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10

0

21 dpi

MOCK-infected C.suis -infected C.trachomatis -infected

Lowest determined concentration in the standard curve Fig. 2. Detection of Cs and Ct in vaginal swabs and horn flushes. (A) Vaginal swabs from MOCK-inoculated (grey circles), Cs-inoculated (green squares) and Ct-inoculated (red triangles) pigs were analysed on chlamydial infection via qPCR prior to infection and at the stated days post inoculation (dpi, x-axis). (B) At necropsy, the uterine horns from the pigs in the three infection groups were flushed with 20 ml PBS. Chlamydial presence in the horn flushes was determined via qPCR. In order to determine the amount of detected chlamydia in swabs and flushes, a standard curve was included in the analysis. The lowest determined concentration in the standard curve (Cq = 38) is depicted as a dashed line. Chlamydia levels below the standard curve should be considered qualitatively. Median chlamydial concentrations are shown as black bars. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.3. Immune cell frequencies in blood, draining lymph nodes and uterine horns

A)

0.4 0.3

The humoral immune responses to chlamydia infection were analysed by ELISA detecting anti-chlamydial MOMP IgG in serum (Fig. 3A) and by analysing serum on the presence of neutralizing antibodies (Fig. 3B). While serum IgG levels were elevated by trend in the Cs- and Ct-inoculated groups at 14 and 21 dpi, the increase never reached significant levels (Fig. 3A). Neutralizing antibody levels in Ct-inoculated pigs were similar to MOCK-inoculated pigs with a minor increase at 14 dpi by trend. In contrast, neutralizing antibody levels in the serum of Cs-inoculated pigs were significantly increased at 14 and 21 dpi (Fig. 3B). 3.5. Proliferative immune response of chlamydia-specific T-cell subsets Isolated PBMC from MOCK-, Cs-, or Ct-inoculated pigs were restimulated with Cs- and Ct-lysates to determine the antigenspecific proliferation of T-cell subsets throughout the study via FCM (Fig. 4). The gating hierarchy to analyse proliferation of CD4+ T cells, CTLs and TCR-cd T cells is illustrated in Supplementary Fig. 4. Starting at 7 dpi Cs-inoculated pigs show a strong and highly significant proliferative response throughout the study upon re-stimulation with both Cs- and Ct-lysate. At 7 and 14 dpi, CD4+ T cells from Ct-inoculated pigs show a proliferative response upon both chlamydial lysate re-stimulations as well, although only the response to Ct lysate is statistically significant while the proliferative response to Cs lysate had p values of 0.056 and 0.059, respectively (Fig. 4, top row). Cytotoxic T lymphocytes showed a very minor proliferative response upon chlamydial re-stimulation with less than 5% proliferating cells (Fig. 4, bottom row). The CTLs from Ct-inoculated pigs seemed to be more responsive to Cs lysate throughout the study compared to MOCK-inoculated pigs, even before inoculation (
2 dpi). In response to Ct lysate re-stimulation, CTLs from Ctinoculated pigs showed a statistically significant proliferation from 7 dpi onwards. Cs-inoculated pigs showed only non-significant CTL proliferation to both chlamydia lysates and only at later time points. The proliferation of TCR-cd T cells was highly variable and did not show any differences between MOCK- and chlamydia-inoculated groups (data not shown). Chlamydia-specific T-cell subset proliferation was also analysed in draining lymph nodes at necropsy (=21 dpi, Fig. 5). Upon re-

OD

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B)

*

50 40

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30 20 10 0 -10 -20 -30 -40

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dp i 14

i dp 7

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3.4. Humoral immune response

0.2

% suppression of infection

Immune cell frequencies in blood were relatively stable across the three infection groups except decreased lymphocyte and monocyte numbers at 3 dpi and elevated levels of monocytes and neutrophils at 21 dpi in the chlamydia-inoculated groups (Supplementary Fig. 2). There were no major differences in the frequencies of the lymphocyte subsets (NK cells, B cells, T cells, CTL, TCR-cd T cells, CD4+ T cells, and Tregs) in blood throughout the study (data not shown). Immune cell frequencies in draining lymph nodes were also very similar between the groups (Supplementary Fig. 3A and B). Within T-cells, the frequencies of TCR-cd T cells, Tregs and experienced CD4+ T cells (CD4+CD8a+) were unaltered by infection. In contrast, Ct-inoculated animals showed a decreased CTL frequency while CD4+ T cells were increased in draining lymph nodes. T-cell frequencies in flushes of the uterine horns from Cs-inoculated pigs were elevated by trend (p = 0.051) while the frequencies of the T-cell subsets were similar in the MOCK- and chlamydia-inoculated pigs. Interestingly, the rate of antigenexperienced cells within CD4+ cells was very high in uterine horns from pigs of all groups with frequencies between 80% and 100% (Supplementary Fig. 3C).

MOCK-infected C.suis -infected C.trachomatis -infected Fig. 3. Humoral immune response to Cs and Ct infection in pigs. The humoral immune response was analysed in MOCK-inoculated (grey circles), Cs-inoculated (green squares) and Ct-inoculated (red triangles) pigs. (A) Anti-chlamydial MOMP serum IgG levels of animals at the depicted days post inoculation (dpi, x-axis) were analysed in comparison to levels before inoculation within the same animal via ELISA (DOD at 1:100 dilution, y-axis). (B) The presence of neutralizing antibodies in porcine serum was analysed by infecting HeLa cells with Cs in the presence of serum (1:10 dilution). The % suppression of infection (y-axis) was calculated by dividing the infection rate at the days post inoculation (dpi, x-axis) by the infection rate before inoculation within the same animal. Data show the average of three experiments. Medians are depicted as black bars. Data were analysed using a repeated-measures 2-way ANOVA in comparison to MOCK-inoculated animals and corrected for multiple comparisons with Dunnett’s multiple comparisons test. * p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

stimulation with Cs lysate, CD4+, CTL and even TCR-cd T cells from Cs-inoculated animals showed a strong and most times significant proliferative response in the iliac and uterine lymph nodes. The proliferative response in draining lymph nodes from Ct-inoculated pigs was less prominent and significant only in the uterine lymph node for CTLs and TCR-cd T cells upon Ct lysate re-stimulation.

3.6. Cytokine response of chlamydia-specific T-cell subsets The chlamydia-specific IFN-c, TNF-a and IL-17 responses of CD4+ T cells, CTLs and TCR-cd T cells were analysed in PBMC via polychromatic FCM (pFCM) upon re-stimulation with Cs- or Ct-lysate. The gating hierarchy used for this pFCM analysis is shown in Supplementary Fig. 5. Fig. 5 shows the frequency of the sum of IFN-c, TNF-a and IL-17 cytokine producers. Before inoculation (
2 dpi) and at 3 dpi, there were no differences in the frequencies of cytokine producing CD4+ T cells, CTLs and TCR-cd T cells between the MOCK- and chlamydia-inoculated groups (data not shown for TCR-cd T cells). At later time points (7–21 dpi), CD4+ T cells from Cs-inoculated pigs showed a strong

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*

*

60

C. trachomatis-lysate re-stimulation

*

+

% proliferating CD4 cells

C. suis-lysate re-stimulation 70

*

*

50

*

40 30

*

*

*

*

20 10 0

% proliferating CTLs

10

* *

*

*

i dp 21

dp i 14

dp i 7

i dp 3

-2

dp i

i dp 21

i dp 14

dp i 7

i dp 3

-2

dp i

0

MOCK-infected C.suis -infected C.trachomatis -infected Fig. 4. Chlamydia-specific proliferative response of T-cell subsets in blood. PBMC were isolated from MOCK-inoculated (grey circles), Cs-inoculated (green squares) and Ct-inoculated (red triangles) pigs before inoculation (
2 dpi) and after inoculation (3–21 dpi) (x-axis). PBMC were stained with CFSE to analyse the proliferation of CD4+ T cells (top row) and cytotoxic T lymphocytes (CTLs, bottom row) after four day in vitro re-stimulation via flow cytometry. Cells were re-stimulated with Cs (left column) or Ct lysate (right column). Medians are depicted as black bars. Data were analysed using repeated-measures 2-way ANOVA in comparison to MOCK-inoculated animals and corrected for multiple comparisons with Dunnett’s multiple comparisons test. * p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and significant cytokine production upon stimulation with both Cs- and Ct-lysate (Fig. 6A, top row). While there was only a trend of IL-17 induction at these time points upon Cs lysate re-stimulation, IFN-c and TNF-a were significantly induced after re-stimulation with both chlamydia lysates (Supplementary Fig. 6). CD4+ T cells from Ct-inoculated pigs showed only a minor and non-significant increase in cytokine production at the later time points (7–21 dpi, Fig. 6A, top row). The cytokine response in CTLs and TCR-cd T cells was very minor and similar between MOCK- and chlamydia-inoculated groups with only a slight and mainly non-significant increased production in Cs-inoculated pigs (Fig. 6A, bottom row, and data not shown).

Cs- (left) or Ct-lysate (right); e.g. if the pie chart includes 50% cytokine-producers, 0.5% of all CD4+ cells were cytokineproducers. CD4+ cells from the five MOCK-inoculated animals (top row) showed consistently a low cytokine production. In contrast, most Cs-inoculated animals (middle row) showed a strong induction of cytokine-producing CD4+ cells while Ct-inoculated animals (bottom row) showed a low to moderate cytokine response. Most cytokine-producing cells in the Cs- and Ct-inoculated animals are either IFN-c single producers (light grey) or IFN-c/TNF-a double-producers (light green) upon both, homologous or heterologous re-stimulation with Cs or Ct lysate. There are only some TNF-a single-producers (intermediate grey) and IFN-c/TNF-a/IL-17 triple-producers (red).

3.7. Induction of multi-functional CD4+ T cells by Cs and Ct 4. Discussion Chlamydia-specific cytokine production of CD4+ T cells in blood from MOCK-, Cs- or Ct-inoculated animals was analysed throughout the study. In contrast to the quantity of the cytokine response, the type of the immune response was similar throughout the study and is shown for 7 dpi in Fig. 6B. While 99% of CD4+ T cells did not produce cytokines, the pie charts show the 1% of cells with production of various amounts of cytokines upon re-stimulation with

The purpose of this study was to decipher the T-cell immune response to chlamydial infections including Ct and Cs with a focus on the most important TH1 immune response. A better understanding of this important immune response will improve the relevance of the pig as a model for developing and testing vaccine candidates against Cs and Ct.

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+

% proliferating CD4 cells

Iliac LN

Uterine LN

*

15 10 5 5

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4 3

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2 1 0

% proliferating CTLs

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C.suis -infected

C.trachomatis -infected

Fig. 5. Chlamydia-specific proliferative response of T-cell subsets in draining lymph nodes. Lymphocytes were isolated from iliac (left) and uterine (right) lymph nodes at necropsy (21 dpi) of MOCK-inoculated (grey circles), Cs-inoculated (green squares) and Ct-inoculated (red triangles) pigs. Cells were stained before culture with CFSE to analyse the proliferation of CD4+ T cells (top row), cytotoxic T lymphocytes (CTLs, middle row), and TCR-cd-T cells (bottom row) upon four day in vitro re-stimulation via flow cytometry. Cells were either not re-stimulated (Medium), or re-stimulated with Cs or Ct lysate (x-axis). Medians are depicted as black bars. Data were analysed using two-way ANOVA in comparison to MOCK-inoculated animals and corrected for multiple comparisons using the Dunnett’s test. * p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Genital infection of pigs with Cs and Ct was performed via intravaginal and intra-uterine inoculation. Cs infections were generally higher and triggered stronger immune responses compared to Ct. The slightly weaker infection using Ct is in line with studies by Vanrompay et al. in Belgium who used a ten times higher infectious dose for their studies on Ct (1  108 TCID50, [6–8,20]) compared to their Cs studies (1  107 TCID50, [21]). Testing the humoral immune response via serum IgG levels and neutralizing antibodies showed that data obtained by the neutralizing antibody test were much clearer and reached a significant level at 14 and 21 dpi after Cs-inoculation. Cs was used to determine the neutralizing antibody levels. This heterologous analysis shows only cross-reactive neutralizing antibodies for Ct inoculated animals. It is therefore possible, that the neutralizing antibody levels for Ct inoculated animals may be higher in a homologous analysis system.

We used polychromatic FCM (pFCM) to combine the analysis of the cellular phenotype with its proliferative or cytokine response upon in vitro re-stimulation with Cs or Ct lysates in order to detect multifunctional T cells which have a great potential on being correlates of protection [22]. In murine C. muridarum infections, CD4+ IFN-c/TNF-a double producers are known to be important immune responders as their abundance has been shown to accurately correlate with protection [13]. The incorporation of pFCM in our study enabled us to show for the first time that CD4+ T cells are main responders to Cs and Ct infection in pigs. Most of these cells were either IFN-c single producers or IFN-c/TNF-a double producers confirming murine studies showing that a TH1 response, inclusive IFN-c/TNF-a double producers, is the main immune response in chlamydia infections and essential for pathogen clearance [11,12,23,24]. These CD4+ IFN-c producers were also associated with a statistically non-significant reduced risk of

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C. suis-lysate re-stimulation

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dp i

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non-producers various

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+TNF

IFN +

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Fig. 6. Cytokine production of chlamydia-specific T-cell subsets in blood. (A) PBMC were isolated from MOCK-inoculated (grey circles), Cs-inoculated (green squares) and Ct-inoculated (red triangles) pigs before inoculation (
2 dpi) and after inoculation (3–21 dpi) (x-axis). PBMC were re-stimulated in vitro for 24 h with Cs (left column) or Ct lysate (right column) in the presence of monensin to block Golgi export for the last 6 h of culture. After in vitro stimulation, cells were harvested and stained for living CD4+ cells (top row) and cytotoxic T lymphocytes (bottom row) in combination with intracellular staining for IFN-c, TNF-a and IL-17. Data shown are the sum of all cytokineproducing cells within the immune cell subset (y-axis). Medians are depicted as black bars. Data were analysed using repeated-measures 2-way ANOVA in comparison to MOCK-inoculated animals and corrected for multiple comparisons using the Dunnett’s test (* p < 0.05). (B) Chlamydia-specific multifunctional CD4+ T cells: PBMC were isolated from 5 MOCK-inoculated (top row), 5 Cs-inoculated (middle row) and 5 Ct-inoculated (bottom row) pigs 7 days post-inoculation. PBMC were re-stimulated in vitro for 24 h with Cs (left) or Ct (right) lysate in the presence of monensin to block Golgi export for the last 6 h of culture. After in vitro stimulation cells were harvested and stained for living CD4+ cells in combination with intracellular staining for IFN-c, TNF-a and IL-17. Explanation at the bottom of the figure: >99% of CD4+ T cells were non-producers, the remaining 1% produced various cytokines (white: non-producers, grey: single producers, green: double producers, red: triple producers). This production is shown in the pie charts of the figure. On average 125,000 CD4+ T cells were included in the analysis (range: 50,000–210,000 cells). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Ct re-infection in humans [25]. IL-17-producing T-cells (TH17 cells) were rare and there was only a trend towards induction of TH17 cells mainly at 7 dpi. The role of TH17 cells in chlamydia infection is not very well understood and is controversially discussed. While some studies suggest that TH17 cells play a role in protection [24], others indicate that they might increase

tissue-damaging [26]. Given the importance of CD4+ IFN-c/TNF-a double producers for the immune response and their role as correlates of protection, and the controversial results on the role of TH17 cells, including multifunctional T-cell analysis via pFCM in vaccine studies represents a major advantage for the porcine chlamydia model.

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Besides the induction of a CD4+ T-cell response, chlamydiaspecific CTLs and TCR-cd T cells could also be detected, mainly in the draining lymph nodes. A more detailed analysis of these local CTL and TCR-cd T cells in pigs might help to elucidate their role in chlamydial infections, which is controversially discussed or barely analysed, respectively [27]. Heterologous in vitro re-stimulation showed that Cs-specific T cells reacted also with Ct lysate. And to a lower extent, cells from Ct-inoculated pigs detected both chlamydia species in in vitro restimulation assays as well. Thus, we could demonstrate that it is possible to potentially design one vaccine for both Cs and Ct. Such a bivalent vaccine would serve a triple purpose – increasing porcine health and decreasing the zoonotic threat by the Cs vaccine, and improving human health by the Ct vaccine – and would represent a prime example for application of the One Health principle. 5. Conclusions In conclusion, both Cs and Ct can infect pigs and cause long-term infections and pathology. Highly sensitive qPCR enables a precise monitoring of the chlamydial load in order to determine any protective effect of vaccine candidates in pigs. Our analyses of the humoral immune response to chlamydial infection include serum IgG levels and a precise and efficient determination of neutralizing antibodies via FCM, and showed a humoral immune response after 14 dpi. The use of pFCM for the analysis of the T-cell-mediated immune response to chlamydia infections enabled a detailed analysis of the most important T-cell-mediated immune response and showed that the TH1 response started at 7 dpi and is also in pigs the most dominant immune response to chlamydial infections. Therefore, due to the combination of a working infection with Cs and Ct, a sensitive infection monitoring and a precise and efficient way of analysing the humoral and T-cell-mediated immune responses, the pig is an excellent animal model for vaccine development against both pathogens at the same time. In addition, it is able to bridge the gap to clinical trials for Ct vaccine candidates with promising results in rodent animal models. Conflict of interest The authors declare no conflict of interests. Authors’ contributions TK, VG and FM planned and organized the study. TK performed the data analysis and drafted the manuscript. TK, JAP, MDO, GH and KL performed the cell isolations as well as the ex vivo and in vitro experiments. TK and JAP assisted the inoculation and necropsy procedure. JE and SW were in charge of animal handling and necropsy. FM and VG supervised the study. All authors read and revised the manuscript and approved its final version. Acknowledgements The manuscript was published with permission of the Director of VIDO as manuscript #785. TK is member of the Vaccine and Edward Jenner Vaccine Society Young Investigator Program (YIP). AP was supported by a Saskatchewan Health Research Foundation (SHRF) Postdoctoral Research Fellowship. This project was supported by an SHRF establishment grant for FM.

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