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Kinetics of cytokine expression in bovine PBMCs and whole blood after in vitro stimulation with foot-and-mouth disease virus (FMDV) antigen
Cytokine 72 (2015) 58–62
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Kinetics of cytokine expression in bovine PBMCs and whole blood after in vitro stimulation with foot-and-mouth disease virus (FMDV) antigen Pervaiz A. Dar a,b,⇑, Irshad A. Hajam a, Velavurthy S. Suryanarayana a, Subodh Kishore a, Ganesh Kondabattula a a b
FMD Research Centre, Indian Veterinary Research Institute (IVRI), Bangalore 560024, India Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Srinagar 190006, India
a r t i c l e
i n f o
Article history: Received 12 October 2014 Received in revised form 1 December 2014 Accepted 3 December 2014
Keywords: Foot-and-mouth disease virus Cattle Interleukin (IL)-2 IL-4 Interferon (IFN)-c
a b s t r a c t The interest in analysing antigen-specific cytokine responses has substantially increased in recent years, in part due to their use in assessing vaccine efficacy. In the present study, the kinetics of IL-2, IL-4 and IFN-c expression was determined in bovine PBMCs by real-time PCR and in whole blood by cytokinerelease assay after in vitro stimulation with recall foot-and-mouth disease virus (FMDV) antigen. The results showed that the cytokine mRNA of IL-2 and IFN-c in PBMCs were induced early (peak induction at 6 h), whereas the IL-4 mRNA showed delayed induction (peaked at 24 h). In contrast, the kinetics of cytokine proteins in whole blood was different and required the accumulation of the proteins before being optimally detected. The peak accumulation of cytokine protein in whole blood was recorded at 72 h for IL-2 and IL-4, and 96 h for IFN-c. The findings of this study are of importance when selecting an optimal time points for measuring antigen-specific cytokine expression in cattle. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Foot-and-mouth disease virus (FMDV) is considered the most important veterinary pathogen because of its highly contagious nature and the devastating effects the virus have on livestock trade and economy. FMDV is a small, non-enveloped single stranded, positive sense RNA virus belonging to genus Apthovirus of Picornaviridae family [1]. FMDV is categorized into seven serotypes (O, A, C, SAT1, SAT2, SAT3 and Asia1) with significant strain differences and subtypes within a given serotype [2]. The immunity against FMDV following natural infection or vaccination is characterized by the development of strong neutralizing antibody response that confers protection against infection with closely matched strains [3]. However, the possible role of T-cell responses against FMDV and the involvement of T-cells in mediating production of neutralizing antibodies are poorly understood. Cytokine profiling, as a measure of T-cell responses, may offer more detailed insights into the mechanisms of protection against FMDV. Among the T-cells cytokines are mainly produced by CD4+ T-cells which have been segregated into two functional subsets,
⇑ Corresponding author at: Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Srinagar 190006, India. Tel.: +91 9796345298; fax: +91 1942262211. E-mail address: [email protected] (P.A. Dar). http://dx.doi.org/10.1016/j.cyto.2014.12.011 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
termed T-helper 1 (Th1) and Th2 cells, based on their production of specific cytokines. Th1 cells are characterized by the production of interferon (IFN)-c and interleukin (IL)-2, whereas Th2 cells produce IL-4 [4,5]. Both Th1 and Th2 cytokines play an important role in the initiation and maintenance of immunity [6,7]. These cytokines often act in an autocrine or paracrine fashion such that they are released and consumed locally at the site of immune response and their level in peripheral blood circulation remain to an undetectable limit [8]. Hence cytokine profiling for immunological monitoring of T-cell responses are best studied by stimulating Tcells in vitro in an antigen specific manner and then analysing the patterns of cytokine induction at mRNA or protein level using various assays, so called antigen-specific cytokine responses. The antigen-specific cytokine response is likely to be an important part of vaccine assessment [9–11] and can be used to monitor immune status of animals during disease progression or following vaccination. To accurately measure antigen-specific cytokine response, it is important to determine the kinetics of cytokine expression which is unique for each cytokine and each antigenic stimuli used. In the present study, as a step toward monitoring antigen-specific cytokine response in cattle against FMDV, we measured in vitro kinetics of cytokine induction in bovine peripheral blood mononuclear cells (PBMCs) and whole blood in response to FMDV recall antigen. As far as we know, there is no previous report in the literature of studies
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P.A. Dar et al. / Cytokine 72 (2015) 58–62
investigating the kinetics of cytokine expression in response to FMDV antigen. We determined the kinetics of IL-2, IL-4 and IFN-c at mRNA level in PBMCs using real-time PCR, and at the protein level in whole blood using cytokine release assay. 2. Material and methods 2.1. FMDV antigen and vaccine FMDV strain O/IND/R2/75 grown in BHK cells was used for inactivated antigen preparation as described elsewhere [12]. The antigen was formulated in Montanide ISA 201 adjuvant as described earlier [13] and the vaccine formulation contained 11.5 lg of the antigen per 2 ml dose. The same antigen was used for in vitro stimulation of PBMCs and whole blood in all assays described in this study. 2.2. Animal vaccination and sampling Male calves aged 6–8 months of Hallikar breed of Indian cattle (Bos indicus) were used in this study. In order to have responding antigen-specific lymphocytes in peripheral blood circulation of animals, three calves (immune animals) were immunized with a single 2 ml dose of the vaccine intramuscularly and three calves (naïve animals) were left unvaccinated. Two weeks post-vaccination blood samples were obtained from the animals and used for the studies described here. Blood samples were drawn from the jugular vein into 10 ml heparin containing Vacutainer tubes (Greiner Bio-One, Austria). The blood samples were carried to the laboratory at ambient temperature (22 °C ± 5 °C) and processed within 3 h of collection. 2.3. PBMC isolation and antigenic stimulation Heparinized blood was diluted 1:1 with PBS (pH 7.4), overlaid onto Histopaque-1077 (Sigma–Aldrich, USA) at 2:1 ratio, and centrifuged at 1,000g for 30–40 min at room temperature. The resulting band of PBMCs was collected, washed three times with PBS, and then suspended in RPMI media (with 10% FCS without antibiotics) at a concentration of 2.5 106 cells/ml. PBMCs isolated from immune or naïve animals were dispensed in a 6-well tissue culture plate (Corning, USA) at 2 ml/well and stimulated with the recall FMDV antigen (10 lg/ml) for various time periods (see Results) in a humidified incubator in 5% CO2 at 37 °C. 2.4. Total RNA extraction and first-strand cDNA synthesis Total RNA was isolated from PBMCs with TRIzol Reagent (Invitrogen, USA) using a single-step RNA isolation protocol prescribed by the manufacturer [14]. Briefly, PBMCs were pelleted and suspended at 5 106 cells in a 250 ll volume of PBS. TRIzol Reagent (0.75 ml) was added to the cell suspension, mixed by pipetting up and down several times, and then left at room temperature for 5 min. Chloroform (0.2 ml) was added to the suspension, vortexed for 15 s and then centrifuged at 12,000g for 15 min at 4 °C. The upper aqueous phase was reclaimed, mixed with 0.5 ml isopropyl alcohol (Sigma) and incubated on ice for 10 min. The RNA was pelleted from samples by centrifugation at 12,000g for 10 min at 4 °C. The RNA pellet was washed twice by adding 1 ml of 75% v/v ethanol in nuclease-free water (Sigma), and centrifugation at 12,000g for 10 min at 4 °C. The RNA pellet was dried partially and finally dissolved in 20 ll of nuclease-free water. The quality and quantity of extracted RNA was monitored on NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The RNA sample with
A280/A260 ratio in range of 1.8–2.0 and A260/A230 ratio in range of 2.0–2.2 were considered pure and used for cDNA synthesis. First-strand cDNA synthesis was carried out on thermal cycler in a 20 ll volume using random hexamer primers and Super Script III First-strand cDNA synthesis kit (Fermentas, USA) as described by the manufacturer.
2.5. Real-time PCR assay The real-time PCR was catalyzed using Power SYBRÒ Green PCR Master Mix (#4367659, Applied Biosystems, USA) and gene specific-primers (Table 1). GAPDH was used as endogenous control against which the different template values were normalized. The assay was initially optimized for amplification conditions, specificity and efficiency. The amplification reactions were carried out in 20 ll reaction volume in a MicroAmpÒ Fast Optical 96 well reaction plates (Applied Biosystems). Each reaction volume contained 10 ll SYBR Master Mix, 1.0 pmol of both forward and reverse primers, and 1 ll of the template cDNA (except in negative controls). The thermal profile used for the reactions was to heat samples for 5 min at 50 °C and 10 min at 95 °C followed by 40 concurrent cycles involving denaturation at 95 °C for 15 s, annealing at 56 °C for 20 s and extension at 72 °C for 30 s on 7500 Real-Time PCR System (Applied Biosystems). All the runs were completed with a melting-curve analysis from 60 °C to 95 °C at 0.1 °C/s to confirm the specificity of amplification and lack of primer dimers. For relative quantification of cytokine mRNA, the comparative Ct method [15] with the following arithmetic formula was used:
Fold induction ¼ EDDCt ¼ EðDCt
sampleDCt CalibratorÞ
where E = Efficiency = 2, if efficiency is 100%. DCt Sample = Ct of cytokine at (t) h – Ct of GADPH at (t) h. DCt Calibrator = Ct of cytokine at (0) h – Ct of GADPH at (0) h. The cytokine levels in all samples are expressed as log10 to the fold induction of mRNA expression relative to the calibrator.
2.6. Whole blood cytokine release assay Blood sample from each animal was dispensed in four 1 ml aliquots in a 24-well tissue culture plate. 100 ll volume of Concavallin A (10 lg/ml in PBS), the FMDV antigen (10 lg/ml) in duplicate and BHK-21 cell lysate (10 lg total protein/ml) was then added to the wells, respectively. The blood cultures were incubated for different time periods (see Results) in a humidified atmosphere under 5% CO2 at 37 °C. After incubation, the plates were centrifuged and supernatant plasmas were harvested from the well, and stored at 20 °C until use. Cytokine levels in the plasma samples were measured using cytokine ELISA kits for bovine IFN-c (#3115-1H-20, MabTech, Sweden), IL-2 (#DY2465, R&D Systems, USA) and IL-4 (#ESS0031, Thermo Scientific) as per the manufacturer’s protocol. The plasmas were used undiluted in 100 ll volume along with the known concentrations of cytokine standards (provided with the kits) in each ELISA plate. The plates were developed and read at optical density (OD) of 450 nm in iMark Microplate Reader (Biorad, USA). The standard curve generated from OD of cytokine standards were used to determine cytokine levels in the samples. Antigen-specific cytokine release is defined as the mean of cytokine levels in FMDV antigen stimulated plasma after subtraction of cytokine level in mock antigen stimulated plasma for that animal.
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Table 1 List of primers specific for bovine cytokines and their characteristics in real-time PCR. Cytokine
Primer sequence
Accession number
Reference (if any)
Primer annealing
Product size
Product Tm (°C)
Amplification efficiency (E)
Correlation coefficient (R2)
IL-2
50 -ACGCGCCCAAGGTTAACGCT-30 / 5-0 TGGGGTTCAAGTTTTTGCTTGGAGC-30 50 -ACCAGCTGATCCCAGTGCTGGTC-30 / 50 -TGCTACAGGCAGCTCCATGCA-30 50 -CTCCGGCCTAACTCTCTCCT-30 / 50 -AGGCCCACCCTTAGCTACAT-30 50 -GGCGTGAACCACGAGAAGTATAA-30 / 50 -CCCTCCACGATGCCAAAGT-30
M13204
–
53 °C
111 bp
75.54
2
0.985
M77120
–
53 °C
149 bp
80.84
1.94
0.994
M29867
–
57 °C
175 bp
77.34
2.02
0.986
U22385
[16]
56 °C
194 bp
81.91
1.98
0.981
IL-4 IFN-c GAPDH
3.3. Kinetics of cytokine protein expression in whole blood
Data were analyzed by an unpaired Student’s t-tests using GraphPad Prism software (San Diego, CA, USA). P < 0.05 was considered statistically significant.
To determine the feasibility of whole blood cytokine release assay to measure the antigen-specific cytokine responses, we cultured whole blood from the immune or naïve animals with 10 lg/ml of the FMDV antigen and the cytokine release was estimated at 0, 6, 12, 24, 48, 72, and 96 h of stimulation. It was observed that FMDV antigen induced the cytokine releases from the whole
For the comparative DCt method (DDCt method) to be valid, the amplification efficiencies of the target gene and the endogenous control must be approximately equal. The amplification efficiency was determined using duplicates of a 5-fold dilution series of the cDNA template and ranged from 1.90 to 2.02 (Table 1). The correlation coefficient (R2) of the amplifications was >0.97, indicating a strong relationship between the detected Ct values and the corresponding relative amount of the template in reactions. The specificity of the amplicon was confirmed by the appearance of a single peak in the melting curve analysis following completion of the amplification reactions.
3.2. Kinetics of cytokine mRNA transcription in PBMCs In order to determine an optimal stimulation time for antigenspecific cytokine expression, we studied the kinetics of IL-2, IL-4 and IFN-c mRNA transcription in bovine PBMCs in response to FMDV recall antigen. PBMCs from immunized or naïve cattle were stimulated with 10 lg/ml of the FMDV antigen, and the cytokine mRNA transcription was assessed at 0, 2, 6, 12 and 24 h using SYBR Green-based real-time PCR assay (Fig. 1). In general, cytokine mRNAs were induced rapidly (2 h) after incubation of PBMCs with FMDV antigen in both immune and naïve animals. However, the pattern of induction varied depending on both the cytokine being measured and immune status of the animal. The antigen-specific IL-2 mRNA transcription was higher at 6–24 h of stimulation in immunized animals as compared to naïve animals. Maximum accumulation of IL-2 mRNA was observed at 6 h of stimulation (p < 0.05) and the transcription decreased afterwards to a non-significant level between 12 and 24 h of stimulation. In contrast, antigen induced IL-4 mRNA transcription was higher from 6 h onwards in immunized animals as compared to naïve animals and the peak induction was observed at 24 h of antigenic stimulation (p < 0.05). The antigen-specific IFN-c mRNA transcription showed a biphasic pattern, the transcription was observed significantly higher (p < 0.05) at 6 h of stimulation in immunized animals as compared to naïve animals which decreased to non-significant level by 12 h and then again increased to significant levels (p < 0.05) at 24 h of stimulation as compared to the naïve animals.
2.5 IL-2 mRNA inducon
*
2
Immune Naive
1.5 1 0.5 0
2
6
12
24
2.5
Fold Inducon ( log 10)
3.1. Optimization of real-time PCR
IL-4 mRNA inducon 2
*
1.5 1 0.5 0
2
6
12
24
2.5
Fold Inducon (log 10)
3. Results
Fold inducon (log 10)
2.7. Statistical analysis
2
IFN-γ mRNA inducon
*
1.5
*
1 0.5 0
2
6
12
24
me (h) Fig. 1. Kinetics of cytokine mRNA transcription from PBMCs stimulated with FMDV antigen. PBMCs from FMDV immune (n = 3) and naïve (n = 3) animals were stimulated with 10 lg FMDV O antigen for 0, 2, 6, 12 and 24 h. RNA was extracted and gene transcription was quantified by real-time PCR. Results are expressed as fold induction (log10) of cytokine mRNA transcription by FMDV antigen stimulated cells compared to the media treated (non-stimulated) cells. GAPDH was used as internal control and mRNA levels at 0 h was used as calibrator. Histograms represent mean cytokine levels and bars represent standard deviation. ⁄p < 0.05 compared to naive animals.
P.A. Dar et al. / Cytokine 72 (2015) 58–62
blood cultures and the release in the immunized cattle was largely higher than the naïve cattle (Fig. 2). The IL-2 and IFN-c release was significantly higher (p < 0.05) from 48 h onwards, whereas, IL-4 release was higher (p < 0.05) from 24 h onwards in immunized animals as compared to the naïve animals. The concentrations of IL-4 accumulation in plasmas of immunized animals were very low as compared to IL-2 and IFN-c concentrations, peak IL-4 release of 200–400 pg/ml was observed at 72 h post-stimulation. The accumulation of IFN-c was highly variable among reactive animals with the peak levels in the range of 500–1300 pg/ml observed at 96 h post-stimulation in immunized cattle. The peak of IL-2 release was observed at 72 h of stimulation and the levels were in the range of 600–1000 pg/ml in immunized animals. 4. Discussion In the present study, we investigated the kinetics of antigen-specific IL-2, IL-4 and IFN-c expression in order to be able to determine the optimal time points for measuring cytokine profiles. The time kinetics seems to be dependent on the type of cytokine, the animal, the recall stimulus and the assay system Immune Naive
1200
IL- 2 pg/ml
1000
IL-2 release
*
*
*
24
48
72
96
*
*
*
*
24
48
72
96
800 600 400 200 0 0
6
12
1200
IL- 4 pg/ml
1000
IL-4 release
800 600 400 200 0 0
6
12
*
1200
IFN-γ release
IFN -γ pg/ml
1000
*
800
*
600 400 200 0 0
6
12
24
48
72
96
Stimulation time (hr) Fig. 2. Kinetics of cytokine release from whole blood cultures stimulated with FMDV antigen. Whole blood from FMDV immune (n = 3) and naïve (n = 3) cattle were stimulated with 10 lg FMDV O antigen for 0, 6,12,24,48,72 and 96 h. Cytokine release was measured in plasmas by ELISA and levels expressed as picogram (pg) per ml. Each data point represents mean cytokine levels of three animals and bar represent standard deviation. ⁄p < 0.05 compared to naive animals.
61
used. Generally, the mRNA detection in PBMCs required a relatively precise timing, whereas the cytokine protein detection in whole blood assay required accumulation of the protein before being detected optimally. A high variability was observed among animals, both at the mRNA and the protein levels, reflecting individual qualitative and quantitative differences. All the cytokine responses were higher in the immunized animals as compared to the naïve animals, indicating that previously primed lymphocytes are able to respond to FMDV recall antigen and contribute to the immunity. The kinetics of cytokine mRNAs and proteins were different as it was expected due to the measurement at different levels and using different assay systems. The cytokine mRNAs of IL-2 and IFN-c showed an early induction (maximum at 6 h). Following the peak induction, IL-2 mRNA decreased rapidly to a non-significant level by 12 h, whereas IFN-c mRNA showed a biphasic pattern, rose again to a significantly higher level (p < 0.05) at 24 h of antigenic stimulation after decreasing at 12 h in immunized animals as compared to the naïve animals. Delayed kinetics was observed for IL-4 mRNA transcript with maximal induction at 24 h of antigenic stimulation in the immunized animals as compared to the naïve animals. In contrast, in whole blood assay all the cytokines accumulated during the culture period with the peak levels recorded at 72 h (IL-2 and IL-4) or 96 h (IFN-c) post-stimulation. The levels of IL-4 protein expression were remarkably low as compared to the IL-2 or IFN-c levels despite the fact that inactivated FMDV antigen predominantly induce Th2 type responses. It is possible that expressions of certain cytokine are affected by factors like binding to a cytokine receptor or consumption due to cell proliferation [17,18] and hence pose difficulty to quantitate in the extracellular medium.The levels of IL-2 in whole blood of immunized animals started to decline whereas IFN-c levels were still increasing by 96 h of antigenic stimulation. The quantitation of cytokines in whole blood is advantageous as it can be rapidly and conveniently processed, and it mimics the in vivo environment. The whole blood more accurately reflects the function of the immune system with cellular interactions preserved in the presence of various plasma proteins having stimulatory or inhibitory effects. Disturbances in the ratio of different cell types is avoided such that animals with lower number of leukocytes remain with a low number in the whole blood culture, which is a potential benefit if the purpose of the measurement is to characterize the immune response of individual animals. In contrast, measurements in PBMCs are disadvantageous since animals with a low number of leukocytes are standardized to the same cell ratio as the other animals before measurement. Cytokine responses (for example, IFN-c) in whole blood assays are routinely measured for the purpose of disease diagnosis in cattle [19–21] and it has been earlier reported that whole blood of cattle produce IFN-c in response to FMDV antigen [11,22]. In summary, the antigen-induced cytokine responses should be measured at appropriate time-points according to their expression kinetics so as to assess cytokine concentrations representative of the peak value. The cytokine quantitation at mRNA levels may be more valuable in a scenario where the cytokine in the extracellular media is difficult to measure (as the case with IL-4) or when the reagents are non-available to measure cytokines at the protein level. Lastly, even though the measurements with the different assays may correlate, it is advisable to determine the kinetics for the particular method that will be used in a study, to make sure that the method employed will provide accurate results. Acknowledgements The authors thank the Director, IVRI, Bareilly and Joint Director, IVRI, Bangalore for providing necessary facilities for carrying out this research work.
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References [1] Grubman MJ, Baxt B. Foot-and-mouth disease. Clin Microbiol Rev 2004;17:465–93. [2] Knowles NJ, Samuel AR. Molecular epidemiology of foot-and-mouth disease virus. Virus Res 2003;91:65–80. [3] Doel TR. Natural and vaccine induced immunity to FMD. Cur Topics Microbiol Immunol 2005;288:103–31. [4] Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two type of murine helper T cell clones. 1. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348–57. [5] Cher DJ, Mosmann TR. Two types of murine helper Tcell clone: II. Delayed-type hypersensitivity is mediated by Th1 clones. J Immunol 1987;138:3688–94. [6] Maldonado-Lopez R, Moser M. Dendritic cell subsets and the regulation of Th1/ Th2 responses. SeminImmunol 2001;13:275–82. [7] Jankovic D, Liu Z, Gause WC. Th1- and Th2-cell commitment during infectious disease: asymmetry in divergent pathways. Trends Immunol 2001;22:450–7. [8] Oppenheim JJ, Feldmann M. Introduction to the role of cytokines in innate host defense and adaptive immunity. Cellular Immunology. New York: Oxford University Press; 2000. [9] Thakur A, Lasse EP, Gregers J. Immune markers and correlates of protection for vaccine induced immune responses. Vaccine 2012;30:4907–20. [10] Plotkin SA. Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis 2008;47:401–9. [11] Parida S, OhY, Reid SM, Cox SJ, Statham RJ, Mahapatra M, et al. Interferongamma production in vitro from whole blood of foot-and-mouth disease virus (FMDV) vaccinated and infected cattle after incubation with inactivated FMDV. Vaccine 2006;24:964–9. [12] Patil PK, Bayry J, Ramakrishna C, Hugar B, Misra LD, Natarajan C. Immune responses of goats against foot-and-mouth disease quadrivalent vaccine: comparison of double oil emulsion and aluminium hydroxide gel vaccines in eliciting immunity. Vaccine 2002;20:2781–9.
[13] Dar P, Kalaivanan R, Sied N, Mamo B, Kishore S, Suryanarayana VVS, et al. Montanide ISA 201 adjuvanted FMD vaccine induces improved immune responses and protection in cattle. Vaccine 2013;31:3327–32. [14] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidiniumthiocyanate–phenol–chloroform extraction. Anal Biochem 1987;162:156–9. [15] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45. [16] Zaros LG, Bricarello PA, Amarante AFT, Coutinho LL. Quantification of bovine cytokine gene expression using real-time RT-PCR methodology. Genet Mol Biol 2007;30:575–9. [17] Collins DP, Luebering BJ, Shaut DM. T-lymphocyte functionality assessed by analysis of cytokine receptor expression, intracellular cytokine expression, and femtomolar detection of cytokine secretion by quantitative flow cytometry. Cytometry 1998;33:249–55. [18] Garrett IR, Durie BG, Nedwin GE, Gillespie A, Bringman T, Sabatini M, et al. Production of lymphotoxin, a bone-resorbing cytokine, by cultured human myeloma cells. N Engl J Med 1987;317:526–32. [19] Stabel JR, Whitlock RH. An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds. Vet immunol immunopathol 2001;79:69–81. [20] Wood PR, Jones SL. BOVIGAM™: an in vitro cellular diagnostic test for bovine tuberculosis. Tuberculosis 2001;81:147–55. [21] Gormley E, Doyle MB, Fitzsimons T, McGill K, Collins JD. Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon (BovigamÒ) assay. Vet Microbiol 2006;112:171–9. [22] Dar PA, Suryanaryana VS, Nagarajan G, Reddy GR, Dechamma HJ, Kondabattula G. DNA prime-protein boost strategy with replicase-based DNA vaccine against foot-and-mouth disease in bovine calves. Vet Microbiol 2013;163:62–70.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Kinetics of cytokine expression in bovine PBMCs and whole blood after in vitro stimulation with foot-and-mouth disease virus (FMDV) antigen Pervaiz A. Dar a,b,⇑, Irshad A. Hajam a, Velavurthy S. Suryanarayana a, Subodh Kishore a, Ganesh Kondabattula a a b
FMD Research Centre, Indian Veterinary Research Institute (IVRI), Bangalore 560024, India Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Srinagar 190006, India
a r t i c l e
i n f o
Article history: Received 12 October 2014 Received in revised form 1 December 2014 Accepted 3 December 2014
Keywords: Foot-and-mouth disease virus Cattle Interleukin (IL)-2 IL-4 Interferon (IFN)-c
a b s t r a c t The interest in analysing antigen-specific cytokine responses has substantially increased in recent years, in part due to their use in assessing vaccine efficacy. In the present study, the kinetics of IL-2, IL-4 and IFN-c expression was determined in bovine PBMCs by real-time PCR and in whole blood by cytokinerelease assay after in vitro stimulation with recall foot-and-mouth disease virus (FMDV) antigen. The results showed that the cytokine mRNA of IL-2 and IFN-c in PBMCs were induced early (peak induction at 6 h), whereas the IL-4 mRNA showed delayed induction (peaked at 24 h). In contrast, the kinetics of cytokine proteins in whole blood was different and required the accumulation of the proteins before being optimally detected. The peak accumulation of cytokine protein in whole blood was recorded at 72 h for IL-2 and IL-4, and 96 h for IFN-c. The findings of this study are of importance when selecting an optimal time points for measuring antigen-specific cytokine expression in cattle. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Foot-and-mouth disease virus (FMDV) is considered the most important veterinary pathogen because of its highly contagious nature and the devastating effects the virus have on livestock trade and economy. FMDV is a small, non-enveloped single stranded, positive sense RNA virus belonging to genus Apthovirus of Picornaviridae family [1]. FMDV is categorized into seven serotypes (O, A, C, SAT1, SAT2, SAT3 and Asia1) with significant strain differences and subtypes within a given serotype [2]. The immunity against FMDV following natural infection or vaccination is characterized by the development of strong neutralizing antibody response that confers protection against infection with closely matched strains [3]. However, the possible role of T-cell responses against FMDV and the involvement of T-cells in mediating production of neutralizing antibodies are poorly understood. Cytokine profiling, as a measure of T-cell responses, may offer more detailed insights into the mechanisms of protection against FMDV. Among the T-cells cytokines are mainly produced by CD4+ T-cells which have been segregated into two functional subsets,
⇑ Corresponding author at: Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Srinagar 190006, India. Tel.: +91 9796345298; fax: +91 1942262211. E-mail address: [email protected] (P.A. Dar). http://dx.doi.org/10.1016/j.cyto.2014.12.011 1043-4666/Ó 2014 Elsevier Ltd. All rights reserved.
termed T-helper 1 (Th1) and Th2 cells, based on their production of specific cytokines. Th1 cells are characterized by the production of interferon (IFN)-c and interleukin (IL)-2, whereas Th2 cells produce IL-4 [4,5]. Both Th1 and Th2 cytokines play an important role in the initiation and maintenance of immunity [6,7]. These cytokines often act in an autocrine or paracrine fashion such that they are released and consumed locally at the site of immune response and their level in peripheral blood circulation remain to an undetectable limit [8]. Hence cytokine profiling for immunological monitoring of T-cell responses are best studied by stimulating Tcells in vitro in an antigen specific manner and then analysing the patterns of cytokine induction at mRNA or protein level using various assays, so called antigen-specific cytokine responses. The antigen-specific cytokine response is likely to be an important part of vaccine assessment [9–11] and can be used to monitor immune status of animals during disease progression or following vaccination. To accurately measure antigen-specific cytokine response, it is important to determine the kinetics of cytokine expression which is unique for each cytokine and each antigenic stimuli used. In the present study, as a step toward monitoring antigen-specific cytokine response in cattle against FMDV, we measured in vitro kinetics of cytokine induction in bovine peripheral blood mononuclear cells (PBMCs) and whole blood in response to FMDV recall antigen. As far as we know, there is no previous report in the literature of studies
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investigating the kinetics of cytokine expression in response to FMDV antigen. We determined the kinetics of IL-2, IL-4 and IFN-c at mRNA level in PBMCs using real-time PCR, and at the protein level in whole blood using cytokine release assay. 2. Material and methods 2.1. FMDV antigen and vaccine FMDV strain O/IND/R2/75 grown in BHK cells was used for inactivated antigen preparation as described elsewhere [12]. The antigen was formulated in Montanide ISA 201 adjuvant as described earlier [13] and the vaccine formulation contained 11.5 lg of the antigen per 2 ml dose. The same antigen was used for in vitro stimulation of PBMCs and whole blood in all assays described in this study. 2.2. Animal vaccination and sampling Male calves aged 6–8 months of Hallikar breed of Indian cattle (Bos indicus) were used in this study. In order to have responding antigen-specific lymphocytes in peripheral blood circulation of animals, three calves (immune animals) were immunized with a single 2 ml dose of the vaccine intramuscularly and three calves (naïve animals) were left unvaccinated. Two weeks post-vaccination blood samples were obtained from the animals and used for the studies described here. Blood samples were drawn from the jugular vein into 10 ml heparin containing Vacutainer tubes (Greiner Bio-One, Austria). The blood samples were carried to the laboratory at ambient temperature (22 °C ± 5 °C) and processed within 3 h of collection. 2.3. PBMC isolation and antigenic stimulation Heparinized blood was diluted 1:1 with PBS (pH 7.4), overlaid onto Histopaque-1077 (Sigma–Aldrich, USA) at 2:1 ratio, and centrifuged at 1,000g for 30–40 min at room temperature. The resulting band of PBMCs was collected, washed three times with PBS, and then suspended in RPMI media (with 10% FCS without antibiotics) at a concentration of 2.5 106 cells/ml. PBMCs isolated from immune or naïve animals were dispensed in a 6-well tissue culture plate (Corning, USA) at 2 ml/well and stimulated with the recall FMDV antigen (10 lg/ml) for various time periods (see Results) in a humidified incubator in 5% CO2 at 37 °C. 2.4. Total RNA extraction and first-strand cDNA synthesis Total RNA was isolated from PBMCs with TRIzol Reagent (Invitrogen, USA) using a single-step RNA isolation protocol prescribed by the manufacturer [14]. Briefly, PBMCs were pelleted and suspended at 5 106 cells in a 250 ll volume of PBS. TRIzol Reagent (0.75 ml) was added to the cell suspension, mixed by pipetting up and down several times, and then left at room temperature for 5 min. Chloroform (0.2 ml) was added to the suspension, vortexed for 15 s and then centrifuged at 12,000g for 15 min at 4 °C. The upper aqueous phase was reclaimed, mixed with 0.5 ml isopropyl alcohol (Sigma) and incubated on ice for 10 min. The RNA was pelleted from samples by centrifugation at 12,000g for 10 min at 4 °C. The RNA pellet was washed twice by adding 1 ml of 75% v/v ethanol in nuclease-free water (Sigma), and centrifugation at 12,000g for 10 min at 4 °C. The RNA pellet was dried partially and finally dissolved in 20 ll of nuclease-free water. The quality and quantity of extracted RNA was monitored on NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The RNA sample with
A280/A260 ratio in range of 1.8–2.0 and A260/A230 ratio in range of 2.0–2.2 were considered pure and used for cDNA synthesis. First-strand cDNA synthesis was carried out on thermal cycler in a 20 ll volume using random hexamer primers and Super Script III First-strand cDNA synthesis kit (Fermentas, USA) as described by the manufacturer.
2.5. Real-time PCR assay The real-time PCR was catalyzed using Power SYBRÒ Green PCR Master Mix (#4367659, Applied Biosystems, USA) and gene specific-primers (Table 1). GAPDH was used as endogenous control against which the different template values were normalized. The assay was initially optimized for amplification conditions, specificity and efficiency. The amplification reactions were carried out in 20 ll reaction volume in a MicroAmpÒ Fast Optical 96 well reaction plates (Applied Biosystems). Each reaction volume contained 10 ll SYBR Master Mix, 1.0 pmol of both forward and reverse primers, and 1 ll of the template cDNA (except in negative controls). The thermal profile used for the reactions was to heat samples for 5 min at 50 °C and 10 min at 95 °C followed by 40 concurrent cycles involving denaturation at 95 °C for 15 s, annealing at 56 °C for 20 s and extension at 72 °C for 30 s on 7500 Real-Time PCR System (Applied Biosystems). All the runs were completed with a melting-curve analysis from 60 °C to 95 °C at 0.1 °C/s to confirm the specificity of amplification and lack of primer dimers. For relative quantification of cytokine mRNA, the comparative Ct method [15] with the following arithmetic formula was used:
Fold induction ¼ EDDCt ¼ EðDCt
sampleDCt CalibratorÞ
where E = Efficiency = 2, if efficiency is 100%. DCt Sample = Ct of cytokine at (t) h – Ct of GADPH at (t) h. DCt Calibrator = Ct of cytokine at (0) h – Ct of GADPH at (0) h. The cytokine levels in all samples are expressed as log10 to the fold induction of mRNA expression relative to the calibrator.
2.6. Whole blood cytokine release assay Blood sample from each animal was dispensed in four 1 ml aliquots in a 24-well tissue culture plate. 100 ll volume of Concavallin A (10 lg/ml in PBS), the FMDV antigen (10 lg/ml) in duplicate and BHK-21 cell lysate (10 lg total protein/ml) was then added to the wells, respectively. The blood cultures were incubated for different time periods (see Results) in a humidified atmosphere under 5% CO2 at 37 °C. After incubation, the plates were centrifuged and supernatant plasmas were harvested from the well, and stored at 20 °C until use. Cytokine levels in the plasma samples were measured using cytokine ELISA kits for bovine IFN-c (#3115-1H-20, MabTech, Sweden), IL-2 (#DY2465, R&D Systems, USA) and IL-4 (#ESS0031, Thermo Scientific) as per the manufacturer’s protocol. The plasmas were used undiluted in 100 ll volume along with the known concentrations of cytokine standards (provided with the kits) in each ELISA plate. The plates were developed and read at optical density (OD) of 450 nm in iMark Microplate Reader (Biorad, USA). The standard curve generated from OD of cytokine standards were used to determine cytokine levels in the samples. Antigen-specific cytokine release is defined as the mean of cytokine levels in FMDV antigen stimulated plasma after subtraction of cytokine level in mock antigen stimulated plasma for that animal.
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Table 1 List of primers specific for bovine cytokines and their characteristics in real-time PCR. Cytokine
Primer sequence
Accession number
Reference (if any)
Primer annealing
Product size
Product Tm (°C)
Amplification efficiency (E)
Correlation coefficient (R2)
IL-2
50 -ACGCGCCCAAGGTTAACGCT-30 / 5-0 TGGGGTTCAAGTTTTTGCTTGGAGC-30 50 -ACCAGCTGATCCCAGTGCTGGTC-30 / 50 -TGCTACAGGCAGCTCCATGCA-30 50 -CTCCGGCCTAACTCTCTCCT-30 / 50 -AGGCCCACCCTTAGCTACAT-30 50 -GGCGTGAACCACGAGAAGTATAA-30 / 50 -CCCTCCACGATGCCAAAGT-30
M13204
–
53 °C
111 bp
75.54
2
0.985
M77120
–
53 °C
149 bp
80.84
1.94
0.994
M29867
–
57 °C
175 bp
77.34
2.02
0.986
U22385
[16]
56 °C
194 bp
81.91
1.98
0.981
IL-4 IFN-c GAPDH
3.3. Kinetics of cytokine protein expression in whole blood
Data were analyzed by an unpaired Student’s t-tests using GraphPad Prism software (San Diego, CA, USA). P < 0.05 was considered statistically significant.
To determine the feasibility of whole blood cytokine release assay to measure the antigen-specific cytokine responses, we cultured whole blood from the immune or naïve animals with 10 lg/ml of the FMDV antigen and the cytokine release was estimated at 0, 6, 12, 24, 48, 72, and 96 h of stimulation. It was observed that FMDV antigen induced the cytokine releases from the whole
For the comparative DCt method (DDCt method) to be valid, the amplification efficiencies of the target gene and the endogenous control must be approximately equal. The amplification efficiency was determined using duplicates of a 5-fold dilution series of the cDNA template and ranged from 1.90 to 2.02 (Table 1). The correlation coefficient (R2) of the amplifications was >0.97, indicating a strong relationship between the detected Ct values and the corresponding relative amount of the template in reactions. The specificity of the amplicon was confirmed by the appearance of a single peak in the melting curve analysis following completion of the amplification reactions.
3.2. Kinetics of cytokine mRNA transcription in PBMCs In order to determine an optimal stimulation time for antigenspecific cytokine expression, we studied the kinetics of IL-2, IL-4 and IFN-c mRNA transcription in bovine PBMCs in response to FMDV recall antigen. PBMCs from immunized or naïve cattle were stimulated with 10 lg/ml of the FMDV antigen, and the cytokine mRNA transcription was assessed at 0, 2, 6, 12 and 24 h using SYBR Green-based real-time PCR assay (Fig. 1). In general, cytokine mRNAs were induced rapidly (2 h) after incubation of PBMCs with FMDV antigen in both immune and naïve animals. However, the pattern of induction varied depending on both the cytokine being measured and immune status of the animal. The antigen-specific IL-2 mRNA transcription was higher at 6–24 h of stimulation in immunized animals as compared to naïve animals. Maximum accumulation of IL-2 mRNA was observed at 6 h of stimulation (p < 0.05) and the transcription decreased afterwards to a non-significant level between 12 and 24 h of stimulation. In contrast, antigen induced IL-4 mRNA transcription was higher from 6 h onwards in immunized animals as compared to naïve animals and the peak induction was observed at 24 h of antigenic stimulation (p < 0.05). The antigen-specific IFN-c mRNA transcription showed a biphasic pattern, the transcription was observed significantly higher (p < 0.05) at 6 h of stimulation in immunized animals as compared to naïve animals which decreased to non-significant level by 12 h and then again increased to significant levels (p < 0.05) at 24 h of stimulation as compared to the naïve animals.
2.5 IL-2 mRNA inducon
*
2
Immune Naive
1.5 1 0.5 0
2
6
12
24
2.5
Fold Inducon ( log 10)
3.1. Optimization of real-time PCR
IL-4 mRNA inducon 2
*
1.5 1 0.5 0
2
6
12
24
2.5
Fold Inducon (log 10)
3. Results
Fold inducon (log 10)
2.7. Statistical analysis
2
IFN-γ mRNA inducon
*
1.5
*
1 0.5 0
2
6
12
24
me (h) Fig. 1. Kinetics of cytokine mRNA transcription from PBMCs stimulated with FMDV antigen. PBMCs from FMDV immune (n = 3) and naïve (n = 3) animals were stimulated with 10 lg FMDV O antigen for 0, 2, 6, 12 and 24 h. RNA was extracted and gene transcription was quantified by real-time PCR. Results are expressed as fold induction (log10) of cytokine mRNA transcription by FMDV antigen stimulated cells compared to the media treated (non-stimulated) cells. GAPDH was used as internal control and mRNA levels at 0 h was used as calibrator. Histograms represent mean cytokine levels and bars represent standard deviation. ⁄p < 0.05 compared to naive animals.
P.A. Dar et al. / Cytokine 72 (2015) 58–62
blood cultures and the release in the immunized cattle was largely higher than the naïve cattle (Fig. 2). The IL-2 and IFN-c release was significantly higher (p < 0.05) from 48 h onwards, whereas, IL-4 release was higher (p < 0.05) from 24 h onwards in immunized animals as compared to the naïve animals. The concentrations of IL-4 accumulation in plasmas of immunized animals were very low as compared to IL-2 and IFN-c concentrations, peak IL-4 release of 200–400 pg/ml was observed at 72 h post-stimulation. The accumulation of IFN-c was highly variable among reactive animals with the peak levels in the range of 500–1300 pg/ml observed at 96 h post-stimulation in immunized cattle. The peak of IL-2 release was observed at 72 h of stimulation and the levels were in the range of 600–1000 pg/ml in immunized animals. 4. Discussion In the present study, we investigated the kinetics of antigen-specific IL-2, IL-4 and IFN-c expression in order to be able to determine the optimal time points for measuring cytokine profiles. The time kinetics seems to be dependent on the type of cytokine, the animal, the recall stimulus and the assay system Immune Naive
1200
IL- 2 pg/ml
1000
IL-2 release
*
*
*
24
48
72
96
*
*
*
*
24
48
72
96
800 600 400 200 0 0
6
12
1200
IL- 4 pg/ml
1000
IL-4 release
800 600 400 200 0 0
6
12
*
1200
IFN-γ release
IFN -γ pg/ml
1000
*
800
*
600 400 200 0 0
6
12
24
48
72
96
Stimulation time (hr) Fig. 2. Kinetics of cytokine release from whole blood cultures stimulated with FMDV antigen. Whole blood from FMDV immune (n = 3) and naïve (n = 3) cattle were stimulated with 10 lg FMDV O antigen for 0, 6,12,24,48,72 and 96 h. Cytokine release was measured in plasmas by ELISA and levels expressed as picogram (pg) per ml. Each data point represents mean cytokine levels of three animals and bar represent standard deviation. ⁄p < 0.05 compared to naive animals.
61
used. Generally, the mRNA detection in PBMCs required a relatively precise timing, whereas the cytokine protein detection in whole blood assay required accumulation of the protein before being detected optimally. A high variability was observed among animals, both at the mRNA and the protein levels, reflecting individual qualitative and quantitative differences. All the cytokine responses were higher in the immunized animals as compared to the naïve animals, indicating that previously primed lymphocytes are able to respond to FMDV recall antigen and contribute to the immunity. The kinetics of cytokine mRNAs and proteins were different as it was expected due to the measurement at different levels and using different assay systems. The cytokine mRNAs of IL-2 and IFN-c showed an early induction (maximum at 6 h). Following the peak induction, IL-2 mRNA decreased rapidly to a non-significant level by 12 h, whereas IFN-c mRNA showed a biphasic pattern, rose again to a significantly higher level (p < 0.05) at 24 h of antigenic stimulation after decreasing at 12 h in immunized animals as compared to the naïve animals. Delayed kinetics was observed for IL-4 mRNA transcript with maximal induction at 24 h of antigenic stimulation in the immunized animals as compared to the naïve animals. In contrast, in whole blood assay all the cytokines accumulated during the culture period with the peak levels recorded at 72 h (IL-2 and IL-4) or 96 h (IFN-c) post-stimulation. The levels of IL-4 protein expression were remarkably low as compared to the IL-2 or IFN-c levels despite the fact that inactivated FMDV antigen predominantly induce Th2 type responses. It is possible that expressions of certain cytokine are affected by factors like binding to a cytokine receptor or consumption due to cell proliferation [17,18] and hence pose difficulty to quantitate in the extracellular medium.The levels of IL-2 in whole blood of immunized animals started to decline whereas IFN-c levels were still increasing by 96 h of antigenic stimulation. The quantitation of cytokines in whole blood is advantageous as it can be rapidly and conveniently processed, and it mimics the in vivo environment. The whole blood more accurately reflects the function of the immune system with cellular interactions preserved in the presence of various plasma proteins having stimulatory or inhibitory effects. Disturbances in the ratio of different cell types is avoided such that animals with lower number of leukocytes remain with a low number in the whole blood culture, which is a potential benefit if the purpose of the measurement is to characterize the immune response of individual animals. In contrast, measurements in PBMCs are disadvantageous since animals with a low number of leukocytes are standardized to the same cell ratio as the other animals before measurement. Cytokine responses (for example, IFN-c) in whole blood assays are routinely measured for the purpose of disease diagnosis in cattle [19–21] and it has been earlier reported that whole blood of cattle produce IFN-c in response to FMDV antigen [11,22]. In summary, the antigen-induced cytokine responses should be measured at appropriate time-points according to their expression kinetics so as to assess cytokine concentrations representative of the peak value. The cytokine quantitation at mRNA levels may be more valuable in a scenario where the cytokine in the extracellular media is difficult to measure (as the case with IL-4) or when the reagents are non-available to measure cytokines at the protein level. Lastly, even though the measurements with the different assays may correlate, it is advisable to determine the kinetics for the particular method that will be used in a study, to make sure that the method employed will provide accurate results. Acknowledgements The authors thank the Director, IVRI, Bareilly and Joint Director, IVRI, Bangalore for providing necessary facilities for carrying out this research work.
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