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Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection
Microbial Pathogenesis 107 (2017) 122e128
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection Changbo Ou a, b, c, 1, Qiuxia Wang a, c, 1, Yan Yu c, Yanhong Zhang c, Jinyou Ma c, Xianghui Kong b, *, Xingyou Liu a, c, ** a b c
Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, Henan, China College of Life Science, Henan Normal University, Xinxiang 453007, Henan, China College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, Henan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2017 Received in revised form 22 March 2017 Accepted 24 March 2017 Available online 27 March 2017
Both CCR5 and CXCR4 are important chemokine receptors and take vital role in migration, development and distribution of T cells, however, whether they will influence the process of T cell infiltration into bursa of Fabricius during infectious bursal disease virus (IBDV) infection is unclear. In the current study, CCR5 and CXCR4 antagonists, Maraviroc and AMD3100, were administrated into chickens inoculated with IBDV, and the gene levels of IBDV VP2, CCR5, CXCR4 and related cytokines were determined by realtime PCR. The results showed that large number of T cells began to migrate into the bursae on Day 3 post infection with IBDV and the mRNA of chemokine receptors CCR5 and CXCR4 began to increase on Day 1. Moreover, antagonist treatments have increased the VP2, CCR5 and CXCR4 gene transcriptions and influenced on the gene levels of IL-2, IL-6, IL-8, IFN-g, TGF-b4, MHC-I and MDA5. In conclusion, the chemokine receptors CCR5 and CXCR4 might influence virus replication during IBDV infection and further study would focus on the interaction between chemokine receptors and their ligands. © 2017 Elsevier Ltd. All rights reserved.
Keywords: CCR5 CXCR4 Infectious bursal disease virus T cell migration
1. Introduction Infectious bursal disease virus (IBDV) is an important small RNA virus in the family of Birnaviridae, which might cause infectious bursal disease (IBD) in young chickens and lead to severe immunosuppressive effects and pathological damage. It mainly targets early B cells, especially those in the gut-associated lymphoid organ, the bursa of Fabricius [1]. Post infection with IBDV, the bursa of Fabricius displays edema and hemorrhage, or even necrosis, accompanying with a large number of T cells infiltration and cytokine storm [2]. However, the key factor which influences T cells migrating to the bursa has not been explored. Moreover, whether the chemokine receptors on the surface of T cell influence the virus replication is also still unclear. Chemokine receptors expressed on the surface of many types of
* Corresponding author. College of Life Science, Henan Normal University, Xinxiang 453007, Henan, China. ** Corresponding author. Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, Henan, China. E-mail addresses: [email protected] (X. Kong), [email protected] (X. Liu). 1 These authors contribute to this manuscript equally. http://dx.doi.org/10.1016/j.micpath.2017.03.031 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
immune cells mediate migration of immune cells to corresponding target organs [3]. CeC chemokine receptor type 5, also known as CCR5 or CD195, is a member of G protein-coupled receptors and involved in the process by which T cells are attracted to specific tissue and target organs. It is specially used as one of HIV invasion receptors and causes great concerns on HIV-1 receptor antagonists [4]. CXCR4, the only one receptor of the Stromal cell Derived Factor 1 (SDF-1, or CXCL12), is one of G protein-coupled receptors and could express on most of tissues and organs, which plays important role in anti-HIV infection, cancer and other human diseases [5,6]. Maraviroc as a new CCR5 antagonist has been reported for shortterm therapy in patients infected with HIV-1 and AMD3100 as a small-molecule antagonist of CXCR4 could alter fracture repair and anticipate in anti-angiogenesis effects in oxygen-induced retinopathy mice [7e9]. Meanwhile, silica analysis of chicken genome has identified 12 chemokine receptors and 10 of them were expressed in T lymphocytes [10,11]. Moreover, Annamalai T has displayed that the mRNA levels of the chemokine receptors, such as CCR7, CCR8, CCR9 and CXCR4, were different in the CD4þ cells isolated from bursa, cecal tonsil and thymus, and the CCR7 and CXCR5 mRNA contents were dependent on the immune organs and the inflammatory status in chickens [12].
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Therefore, though both of CCR5 and CXCR4 are important receptors and take part in migration, development and distribution of T cells, how they will take part in the process of T cell infiltration into bursa of Fabricius during IBDV infection is unclear [13,14]. This study would determine the relative levels of mRNA of CCR5 and CXCR4 in bursa and the effects of their antagonists on IBDV infection. We aimed to explore which chemokine receptors would take part in T cell migration during IBDV infection and know whether chemokine receptors blockade would influence the virus replication. 2. Material and methods 2.1. Antagonists, viruses and birds Maraviroc (Sigma-Aldrich, MO, USA) and AMD3100 (Abcam, UK) were purchased by the commercial company. A classical IBDV strain CJ801 was kindly provided by Prof. Jue Liu, Beijing Academy of Agriculture and Forestry, Beijing, China. All the studies were performed on 3-week-old specific-pathogen-free (SPF) chickens (Vital Bridge Co. Ltd., Beijing, China). The experimental procedures and animal management were approved by the Institutional Animal Care and Use Committee at Henan Institute of Science and Technology. 2.2. Experiment design 2.2.1. Experiment one Eighteen 21-day-old SPF white leghorn chickens were randomly divided into two groups with nine for each group: the mock group (the healthy group) and the IBDV-inoculated group (the infection group). Chickens from the infection group were inoculated with IBDV CJ801 stock containing a dose of 105.24 EID50/0.1 mL through the eye-nose drop [15,16]. The mock group were kept in a separate isolator and mock challenged with PBS. On day 1 and 3 after infection, three chickens from each group were killed under anesthesia for bursa collection. Each bursa was divided into two equal parts: one part for T cell counts by flow cytometry, the other part was immediately put into liquid nitrogen and then stored in 80 C refrigerator for RNA extraction and chemokine receptors determination by real-time PCR. 2.2.2. Experiment two Twenty-four 21-day-old SPF white leghorn chicken were randomly divided into four groups with six for each group: the mock group (the healthy group), the IBDV-inoculated group (the infection group), the CCR5 antagonist-treated group (Maraviroc group) and CXCR4 antagonist-treated group (AMD3100 group). The mock group was inoculated with physiological saline through the eye-nose drop while the other three groups were inoculated with IBDV CJ801 stock at a dose of 105.24 EID50/0.1 mL through the eyenose drop [15,16]. Based on the previous studies on biological concentrations, Maraviroc and AMD3100 were dissolved in distilled water and orally fed two days before IBDV infection at the dose of 0.5 and 1.0 mg/kg body weight per day, respectively [8,17]. The antagonists were fed once daily for five days until the end of the experiment. After the autopsy, each bursa was immediately put into liquid nitrogen and then stored in 80 C refrigerator for IBDV VP2 genes and cytokine genes expressions.
following the manufacturer's introductions. The cDNA was then analyzed for CCR5, CXCR4, IBDV VP2 and related genes mRNA levels using b-actin as reference gene by the real-time PCR. IL-2, IL-6 and IL-8 are important inflammatory chemotactic factors. IFN-g, MDA5 and TFG-b4 are over-expressed during IBDV infection and play important roles in host antiviral responses. MHC-I molecule is to display intracellular protein to cytotoxic T cells. So IL-2, IL-6, IL-8, IFN-g, TGF-b4, MHC-I and MDA5 are selected for evaluating the effects of antagonists treatment. The primers for these genes are listed in Table 1. The PCR was performed using a 7300 Real-Time PCR System (ABI, Britain) with SYBR® Select Master Mix (ThermoFisher Scientific, China). The reaction conditions were 30 s at 95 C, 40 cycles of 5 s at 95 C, 25 s at 58 C and 30 s at 72 C, followed by a final step of melting curve analysis. After amplification, the relative fold change of the differentially expressed genes was calculated through the 2DDCT method [20]. All samples were performed in two or three replicates to make sure the reproducibility of the amplification. 2.4. Analysis of T-lymphocyte subsets The bursas were collected to detect the relative amounts of CD3þT cells as previously described [16,21]. Briefly, the bursa was ground into cell suspension and then 106 cells were incubated with mouse anti-chicken CD3-SPRD, (Southern Biotech, Birmingham, AL) at 4 C for 30 min. Next, the cells were washed 3 times with phosphate-buffered saline containing 1% fetal bovine serum and then analyzed by the FacsCalibur and CellQuest software (Becton Dickinson, Franklin Lakes, NJ). The viable lymphocytes were calculated on the basis of forward and sideward scatter characteristics, and 20,000 events were analyzed for positive staining with SPRD antibodies. 3. Statistical analysis All data were analyzed through the software package SPSS 13.0 for Windows. Results were displayed as mean ± standard deviation (SD). A two-tailed one-way ANOVA was used to examine the difference between the infection group and the mock group for all statistical analysis.
Table 1 Genes and primers for real-time PCR used in the text. Primer
Sequence
Reference
CCR5
Sense: 5-CTGCCCATGACTGGATTT-3 Anti-sense: 5-GCTGGTGAGGATGCCGTA-3 Sense: 5-TCTTGCCAACCATCTACTCC-3 Anti-sense: 5-ATGACATGAACTGCCTTACA-3 Sense: 5-AGACCCCATTCCCGCTAT-3 Anti-sense: 5-GCCTTGGACGCTTGTTTG-3 Sense: 5- GTACTGATCTTTGGCTGTATTTCGG-3 Anti-sense: 5-CCTGGGTCTCAGTTGGTGTGTAG-3 Sense: 5-AAATCCCTCCTCGCCAATCTG-3 Anti-sense: 5-CCTCACGGTCTTCTCCATAAACG-3 Sense: 5-GCTGCTCTGTCGCAAGGTAGG-3 Anti-sense: 5-ACATCTTGAATGGATTTAGGGTGG-3 Sense: 5-ATCATACTGAGCCAGATTGTTTCG-3 Anti-sense: 5-CTTTCACCTTCTTCACGCCATC-3 Sense: 5-TGCGTCCTCTTTACATTGACTTCC-3 Anti-sense: 5-CGTGCTGTGAGTGGTGTCTGC-3 Sense: 5-CTTCATTGCCTTCGACAAAG-3 Anti-sense: 5-GCCACTCCACGCAGGT-3 Sense: 5-TGAAGGCAAAGAGAGATCAGCGTAAGA-3 Anti-sense:5-CATATCAATTGTGGCAATTCTTGCACAGGA-3 Sense: 5-TTCACCACCACAGCCGAGAG-3 Anti-sense: 5-ACCACAGGACTCCATACCCAAG-3
e
CXCR4 VP2 IL-2 IL-6 IL-8 IFN-g TGF-b4 MHC-I
2.3. RNA isolation and quantitative real-time PCR Total RNA was extracted from chicken bursas by total RNA Kit (Omega Bio-Tek, Guangzhou, China) and then reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, CA, USA)
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MDA5 Actin
e indicates no reference.
e e e e e e e [18] [19] e
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4. Results
expressions were earlier than T cells infiltration.
4.1. Chemokine receptors gene expressions were earlier than T cells infiltration
4.2. CCR5 and CXCR4 antagonist treatments increased IBDV VP2 mRNA levels in bursa
The relationship between T cell infiltration into the bursae and chemokine receptors gene expression was displayed in Fig. 1. CD3þ T cells began to largely home into the bursae on Day 3 after infection with IBDV and increased to 29.4%. However, the mRNA of chemokine receptors, including CCR5 and CXCR4, in the surface of T cells began to increase only on Day 1 and then largely decreased to 5.1 and 0.13% on Day 3. In all, chemokine receptors gene
Post infection with IBDV, some clinical symptoms, such as decreased feed intake, fluffed feathers and diarrhea, were seen in chickens from the infected group. Pathological changes displayed that the bursa of Fabricius was hemorrhage and necrosis. Moreover, chickens from the Maraviroc-treated group and the AMD3100treated group demonstrated similar clinical symptoms with the infected group; some symptoms were even more serious. To
Fig. 1. Relationships between T cell infiltration and relative mRNA levels of CCR5 and CXCR4 in bursae by real-time PCR (n ¼ 3). (A) Total T cell percentage in burase; (B) Relative mRNA levels (mean ± SD) of chemokine receptor CCR5 in the bursa of Fabricius; (C) Relative mRNA levels (mean ± SD) of chemokine receptor CXCR4 in the bursa of Fabricius. ** Indicates P < 0.01 the infection group vs the mock group on Day 1 or Day 3 post-inoculation with IBDV.
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Fig. 2. Quantification of the infectious bursal disease virus (IBDV) VP2 gene levels the bursa of Fabricius in the experimental groups [the mock, infection, Maraviroc and AMD3100 group] using real-time PCR. The data were expressed as the average ratio of IBDV/b-actin. **Indicates P < 0.01 the infection group vs the chemokine receptor antagonist-treatment groups.
determine the effects of chemokine receptors antagonists on IBDV replication in bursa, IBDV VP2 genes in the bursa from the IBDVinfected group were quantified by real-time PCR on day 3 postinfection (Fig. 2). The results showed that VP2 gene expressions of the chemokine receptors antagonists-treated groups were two to three times higher than that of the infected group.
significantly higher than that of the mock group. Moreover, the CCR5 gene expressions of the Maraviroc-treated group and the AMD3100-treated group were approximately 5-fold and 3-fold higher than the CCR5 mRNA level of the infection group, respectively. However, IBDV infection induced the lower CXCR4 mRNA levels of the infection group than the mock group while the CXCR4 gene levels of the Maraviroc-treated group and the AMD3100treated group almost returned to the normal level.
4.3. Antagonist treatments did not decrease chemokine receptors CCR5 and CXCR4 mRNA levels of in bursa The mRNA contents of CCR5 and CXCR4 of bursa on day 3 post infection were quantified by real-time PCR and displayed different expression trends (Fig. 3). After inoculation with IBDV, the CCR5 mRNA levels of the bursa from the IBDV-infected groups were
4.4. Antagonist treatments influenced related cytokines mRNA expressions in bursa To explore why antagonist treatments increase IBDV VP2 gene levels, some related cytokines gene expressions were shown in
Fig. 3. Relative mRNA levels (mean þ SD) of chemokine receptors (A) CCR5 and (B) CXCR4 in the bursa of Fabricius (n ¼ 3). The mRNA levels were determined by quantitative realtime PCR analysis and were normalized to b-actin. **Indicates P < 0.01 when the infection group was compared with the chemokine receptor antagonist-treatment groups. # Indicates P < 0.05 when the AMD3100 group was compared with the Maraviroc group. ##Indicates P < 0.01 when the AMD3100 group was compared with the Maraviroc group.
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Fig. 4. The effect of Maraviroc and AMD3100 treatments on the levels of IL-2 (A), IFN-g (B), IL-6 (C), IL-8 (D), TGF-b4 (E), MHC-I (F) and MDA5 (G) in the bursa of Fabricius by realtime PCR on day 3 post infection with IBDV. **Indicates P < 0.01 when the infection group was compared with the chemokine receptor antagonist-treatment groups. ##Indicates P < 0.01 when the AMD3100 group was compared with the Maraviroc group. “c”Indicates P < 0.01 when the Maraviroc group was compared with the infection and AMD3100 groups.
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Fig. 4. Maraviroc and AMD3100 could significantly induce higher gene transcriptions of IL-2, IL-6, TGF-b4 and MHC-I compared with the infection group (P < 0.01). Moreover, the effects of Maraviroc on cytokines regulation, such as IL-2, IL-6, TGF-b4, MHC-I and MDA5, were better than AMD3100 did (P < 0.01). However, antagonist treatments did not influence the gene levels of IFN-g during IBDV infection, or even worse for IL-8 levels. 5. Discussions Normal and healthy chickens almost have no resident bursal T cell, however, lots of T cells infiltrated into the bursae accompanied with IBDV replication at the site of virus replication [21]. Influx of T cells modulated IBDV pathogenesis by limiting viral replication, promoting tissue damage and delaying tissue recovery [22]. However, the key factors which influenced T cell infiltration have not been known. Chemokine receptors played an important role in migrating T cells into different organs during health and disease development. CCR5 and CXCR4 have been reported to take part in migration, development and distribution of T cells and the present study has also demonstrated that both CCR5 and CXCR4 took part in the process of T cell migration during IBDV infection. Feeding CCR5 and CXCR4 antagonists could increase IBDV VP2 gene expression level and regulate cytokines expression levels induced by T cells. T cells have been shown to limit viral replication in the bursa in the early phase of IBDV infection; therefore, in other words, we speculated that T cell migration delay could increase virus replication in the early phase. Maraviroc is a CCR5 chemokine receptor antagonist while AMD3100 is used to be a highly selective CXCR4 receptor antagonist and potently inhibit SDF-1 binding [23e25]. Our study has demonstrated these antagonists have promoted virus replication on day 3 post inoculation with IBDV, of which the results verified our speculation. Actually, CCR5 has already been reported to be a host factor for Dengue virus-2 replication and disease development and CCR5 antagonist treatment led to a decrease in Dengue virus-2 titres throughout all time-points [17]. Moreover, immune cell recruitment into liver of HBx-TG mice was blocked by a CXCR4 antagonist [26]. These results indicated that blockade of some important chemokine receptor exerted substantial role on virus replication and disease development. Influx of T cells into the bursa induced high expressions levels of CCR5 and low gene content of CXCR4 in the bursa in the present study, which was very interesting. Both CCR5 and CXCR4 were the important chemokine receptor expressed on the surface of T cells, why did they display the different gene expression levels? Moreover, feeding antagonists could increase CCR5 gene levels and offset a downward trend of CXCR4 gene levels in bursa from chickens inoculated with IBDV. We believed that excessive T cells migration into bursa induced high CCR5 gene expressions, though the T cells could not inhibit IBDV reproduction after chemokine receptor antagonist administration. Meanwhile, CXCR4 gene levels only returned to the normal level after chemokine receptor antagonist treatment due to the possibility of inhibiting CXCR4 gene expressions induced by IBDV infection. The CCR5 expression was severalfold increase in the inflammatory cerebrospinal fluid in the process of flavivirus encephalitis, but CCR5 expression decreased in the CCR5D32/wt genotype patients without a compensatory increase of its ligand CCL5 expression, indicating CCR5 expression in the activated lymphocyte population is influenced during flavivirus encephalitis [27]. Previous study has demonstrated that the CXCL12/ CXCR4 pathway exerted huge effects in human papillomavirusinduced pathogenesis and selectively blockade of this axis was a potentially treatment option, of which the results were inconsistent with our results [28]. Therefore, during the progress of IBDV infection and T cell migration, CCR5 and CXCR4 played a different
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role and further study should be focused on the mechanisms of their different functions. In order to explain why virus replication was strengthened after antagonist treatment, some cytokines and immune-related genes were determined by qRT-PCR. Gene transcription of proinflammatory cytokines IL-6 and IL-8 exhibits up-regulation during IBDV infection [29]. However, the present study revealed that antagonist treatments could elevate IL-6 gene levels and decrease IL-8 gene level compared with the infection group. Th1 cytokines IL-2 and IFN-g was also significantly increased to activate T cells in the process of IBDV infection, similarly, administration with Maraviroc and AMD3100 also induced higher levels of Th1 cytokines than the infection group [30]. Anti-inflammatory cytokine TGF-b4, antiviral activation genes MHC-I and MDA5 as one member of RIGI-like receptors were all promoted gene expressions after antagonist treatment compared with the infection group [19,29]. Chemokine receptor antagonists could elevate gene transcriptions of these pro-inflammatory cytokine, Th1 cytokines, antiinflammatory cytokine and other related genes except IL-8 compared with the infection group; meanwhile, the effects of Maraviroc seemed to be more effective than AMD3100 did, which might explain why the virus loads in the Maraviroc-treated group were higher than those of the AMD3100-treated group. Author's contribution OC and WQ designed the experiment and drafted the manuscript. YZ and YY participated in the experiment and bioinformatics analysis of the sequencing data. YY and MJ performed the molecular-related experiments. LX and KX participated in the design of the experiment, the discussion and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (No.31402169 and 31602024), China Postdoctoral Science Foundation funded project (No.2015M570630), Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 13HASTIT006), Key scientific research projects of Henan Province (No.15A230002) and post doctoral fund of Henan institute of science and technology. References [1] T. Ruby, C. Whittaker, D.R. Withers, M.K. Chelbi-Alix, V. Morin, A. Oudin, J.R. Young, R. Zoorob, Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection, J. Virology 80 (18) (2006) 9207e9216. [2] F. Rauw, B. Lambrecht, T. van den Berg, Pivotal role of ChIFNgamma in the pathogenesis and immunosuppression of infectious bursal disease, Avian Pathology J. WVPA 36 (5) (2007) 367e374. [3] B. Homey, A. Muller, A. Zlotnik, Chemokines: agents for the immunotherapy of cancer? Nat. Rev. Immunol. 2 (3) (2002) 175e184. [4] S. Serrano-Villar, T. Sainz, Z.M. Ma, N.S. Utay, T. Wook-Chun, S. Mann, A.D. Kashuba, B. Siewe, A. Albanese, P. Troia-Cancio, et al., Effects of combined CCR5/integrase inhibitors-based regimen on mucosal immunity in HIVinfected patients naive to antiretroviral therapy: a pilot randomized trial, PLoS Pathog. 12 (1) (2016) e1005381. [5] B. Debnath, S. Xu, F. Grande, A. Garofalo, N. Neamati, Small molecule inhibitors of CXCR4, Theranostics 3 (1) (2013) 47e75. [6] S. Oishi, N. Fujii, Peptide and peptidomimetic ligands for CXC chemokine receptor 4 (CXCR4), Org. Biomol. Chem. 10 (30) (2012) 5720e5731. [7] G. Fatkenheuer, A.L. Pozniak, M.A. Johnson, A. Plettenberg, S. Staszewski, A.I. Hoepelman, M.S. Saag, F.D. Goebel, J.K. Rockstroh, B.J. Dezube, et al., Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1, Nat. Med. 11 (11) (2005) 1170e1172. [8] C.A. Toupadakis, A. Wong, D.C. Genetos, D.J. Chung, D. Murugesh, M.J. Anderson, G.G. Loots, B.A. Christiansen, A.S. Kapatkin, C.E. Yellowley, Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair, J. Orthop. Res. Official Publ. Orthop. Res. Soc.
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection Changbo Ou a, b, c, 1, Qiuxia Wang a, c, 1, Yan Yu c, Yanhong Zhang c, Jinyou Ma c, Xianghui Kong b, *, Xingyou Liu a, c, ** a b c
Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, Henan, China College of Life Science, Henan Normal University, Xinxiang 453007, Henan, China College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, Henan, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 February 2017 Received in revised form 22 March 2017 Accepted 24 March 2017 Available online 27 March 2017
Both CCR5 and CXCR4 are important chemokine receptors and take vital role in migration, development and distribution of T cells, however, whether they will influence the process of T cell infiltration into bursa of Fabricius during infectious bursal disease virus (IBDV) infection is unclear. In the current study, CCR5 and CXCR4 antagonists, Maraviroc and AMD3100, were administrated into chickens inoculated with IBDV, and the gene levels of IBDV VP2, CCR5, CXCR4 and related cytokines were determined by realtime PCR. The results showed that large number of T cells began to migrate into the bursae on Day 3 post infection with IBDV and the mRNA of chemokine receptors CCR5 and CXCR4 began to increase on Day 1. Moreover, antagonist treatments have increased the VP2, CCR5 and CXCR4 gene transcriptions and influenced on the gene levels of IL-2, IL-6, IL-8, IFN-g, TGF-b4, MHC-I and MDA5. In conclusion, the chemokine receptors CCR5 and CXCR4 might influence virus replication during IBDV infection and further study would focus on the interaction between chemokine receptors and their ligands. © 2017 Elsevier Ltd. All rights reserved.
Keywords: CCR5 CXCR4 Infectious bursal disease virus T cell migration
1. Introduction Infectious bursal disease virus (IBDV) is an important small RNA virus in the family of Birnaviridae, which might cause infectious bursal disease (IBD) in young chickens and lead to severe immunosuppressive effects and pathological damage. It mainly targets early B cells, especially those in the gut-associated lymphoid organ, the bursa of Fabricius [1]. Post infection with IBDV, the bursa of Fabricius displays edema and hemorrhage, or even necrosis, accompanying with a large number of T cells infiltration and cytokine storm [2]. However, the key factor which influences T cells migrating to the bursa has not been explored. Moreover, whether the chemokine receptors on the surface of T cell influence the virus replication is also still unclear. Chemokine receptors expressed on the surface of many types of
* Corresponding author. College of Life Science, Henan Normal University, Xinxiang 453007, Henan, China. ** Corresponding author. Postdoctoral Research Base, Henan Institute of Science and Technology, Xinxiang 453003, Henan, China. E-mail addresses: [email protected] (X. Kong), [email protected] (X. Liu). 1 These authors contribute to this manuscript equally. http://dx.doi.org/10.1016/j.micpath.2017.03.031 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
immune cells mediate migration of immune cells to corresponding target organs [3]. CeC chemokine receptor type 5, also known as CCR5 or CD195, is a member of G protein-coupled receptors and involved in the process by which T cells are attracted to specific tissue and target organs. It is specially used as one of HIV invasion receptors and causes great concerns on HIV-1 receptor antagonists [4]. CXCR4, the only one receptor of the Stromal cell Derived Factor 1 (SDF-1, or CXCL12), is one of G protein-coupled receptors and could express on most of tissues and organs, which plays important role in anti-HIV infection, cancer and other human diseases [5,6]. Maraviroc as a new CCR5 antagonist has been reported for shortterm therapy in patients infected with HIV-1 and AMD3100 as a small-molecule antagonist of CXCR4 could alter fracture repair and anticipate in anti-angiogenesis effects in oxygen-induced retinopathy mice [7e9]. Meanwhile, silica analysis of chicken genome has identified 12 chemokine receptors and 10 of them were expressed in T lymphocytes [10,11]. Moreover, Annamalai T has displayed that the mRNA levels of the chemokine receptors, such as CCR7, CCR8, CCR9 and CXCR4, were different in the CD4þ cells isolated from bursa, cecal tonsil and thymus, and the CCR7 and CXCR5 mRNA contents were dependent on the immune organs and the inflammatory status in chickens [12].
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Therefore, though both of CCR5 and CXCR4 are important receptors and take part in migration, development and distribution of T cells, how they will take part in the process of T cell infiltration into bursa of Fabricius during IBDV infection is unclear [13,14]. This study would determine the relative levels of mRNA of CCR5 and CXCR4 in bursa and the effects of their antagonists on IBDV infection. We aimed to explore which chemokine receptors would take part in T cell migration during IBDV infection and know whether chemokine receptors blockade would influence the virus replication. 2. Material and methods 2.1. Antagonists, viruses and birds Maraviroc (Sigma-Aldrich, MO, USA) and AMD3100 (Abcam, UK) were purchased by the commercial company. A classical IBDV strain CJ801 was kindly provided by Prof. Jue Liu, Beijing Academy of Agriculture and Forestry, Beijing, China. All the studies were performed on 3-week-old specific-pathogen-free (SPF) chickens (Vital Bridge Co. Ltd., Beijing, China). The experimental procedures and animal management were approved by the Institutional Animal Care and Use Committee at Henan Institute of Science and Technology. 2.2. Experiment design 2.2.1. Experiment one Eighteen 21-day-old SPF white leghorn chickens were randomly divided into two groups with nine for each group: the mock group (the healthy group) and the IBDV-inoculated group (the infection group). Chickens from the infection group were inoculated with IBDV CJ801 stock containing a dose of 105.24 EID50/0.1 mL through the eye-nose drop [15,16]. The mock group were kept in a separate isolator and mock challenged with PBS. On day 1 and 3 after infection, three chickens from each group were killed under anesthesia for bursa collection. Each bursa was divided into two equal parts: one part for T cell counts by flow cytometry, the other part was immediately put into liquid nitrogen and then stored in 80 C refrigerator for RNA extraction and chemokine receptors determination by real-time PCR. 2.2.2. Experiment two Twenty-four 21-day-old SPF white leghorn chicken were randomly divided into four groups with six for each group: the mock group (the healthy group), the IBDV-inoculated group (the infection group), the CCR5 antagonist-treated group (Maraviroc group) and CXCR4 antagonist-treated group (AMD3100 group). The mock group was inoculated with physiological saline through the eye-nose drop while the other three groups were inoculated with IBDV CJ801 stock at a dose of 105.24 EID50/0.1 mL through the eyenose drop [15,16]. Based on the previous studies on biological concentrations, Maraviroc and AMD3100 were dissolved in distilled water and orally fed two days before IBDV infection at the dose of 0.5 and 1.0 mg/kg body weight per day, respectively [8,17]. The antagonists were fed once daily for five days until the end of the experiment. After the autopsy, each bursa was immediately put into liquid nitrogen and then stored in 80 C refrigerator for IBDV VP2 genes and cytokine genes expressions.
following the manufacturer's introductions. The cDNA was then analyzed for CCR5, CXCR4, IBDV VP2 and related genes mRNA levels using b-actin as reference gene by the real-time PCR. IL-2, IL-6 and IL-8 are important inflammatory chemotactic factors. IFN-g, MDA5 and TFG-b4 are over-expressed during IBDV infection and play important roles in host antiviral responses. MHC-I molecule is to display intracellular protein to cytotoxic T cells. So IL-2, IL-6, IL-8, IFN-g, TGF-b4, MHC-I and MDA5 are selected for evaluating the effects of antagonists treatment. The primers for these genes are listed in Table 1. The PCR was performed using a 7300 Real-Time PCR System (ABI, Britain) with SYBR® Select Master Mix (ThermoFisher Scientific, China). The reaction conditions were 30 s at 95 C, 40 cycles of 5 s at 95 C, 25 s at 58 C and 30 s at 72 C, followed by a final step of melting curve analysis. After amplification, the relative fold change of the differentially expressed genes was calculated through the 2DDCT method [20]. All samples were performed in two or three replicates to make sure the reproducibility of the amplification. 2.4. Analysis of T-lymphocyte subsets The bursas were collected to detect the relative amounts of CD3þT cells as previously described [16,21]. Briefly, the bursa was ground into cell suspension and then 106 cells were incubated with mouse anti-chicken CD3-SPRD, (Southern Biotech, Birmingham, AL) at 4 C for 30 min. Next, the cells were washed 3 times with phosphate-buffered saline containing 1% fetal bovine serum and then analyzed by the FacsCalibur and CellQuest software (Becton Dickinson, Franklin Lakes, NJ). The viable lymphocytes were calculated on the basis of forward and sideward scatter characteristics, and 20,000 events were analyzed for positive staining with SPRD antibodies. 3. Statistical analysis All data were analyzed through the software package SPSS 13.0 for Windows. Results were displayed as mean ± standard deviation (SD). A two-tailed one-way ANOVA was used to examine the difference between the infection group and the mock group for all statistical analysis.
Table 1 Genes and primers for real-time PCR used in the text. Primer
Sequence
Reference
CCR5
Sense: 5-CTGCCCATGACTGGATTT-3 Anti-sense: 5-GCTGGTGAGGATGCCGTA-3 Sense: 5-TCTTGCCAACCATCTACTCC-3 Anti-sense: 5-ATGACATGAACTGCCTTACA-3 Sense: 5-AGACCCCATTCCCGCTAT-3 Anti-sense: 5-GCCTTGGACGCTTGTTTG-3 Sense: 5- GTACTGATCTTTGGCTGTATTTCGG-3 Anti-sense: 5-CCTGGGTCTCAGTTGGTGTGTAG-3 Sense: 5-AAATCCCTCCTCGCCAATCTG-3 Anti-sense: 5-CCTCACGGTCTTCTCCATAAACG-3 Sense: 5-GCTGCTCTGTCGCAAGGTAGG-3 Anti-sense: 5-ACATCTTGAATGGATTTAGGGTGG-3 Sense: 5-ATCATACTGAGCCAGATTGTTTCG-3 Anti-sense: 5-CTTTCACCTTCTTCACGCCATC-3 Sense: 5-TGCGTCCTCTTTACATTGACTTCC-3 Anti-sense: 5-CGTGCTGTGAGTGGTGTCTGC-3 Sense: 5-CTTCATTGCCTTCGACAAAG-3 Anti-sense: 5-GCCACTCCACGCAGGT-3 Sense: 5-TGAAGGCAAAGAGAGATCAGCGTAAGA-3 Anti-sense:5-CATATCAATTGTGGCAATTCTTGCACAGGA-3 Sense: 5-TTCACCACCACAGCCGAGAG-3 Anti-sense: 5-ACCACAGGACTCCATACCCAAG-3
e
CXCR4 VP2 IL-2 IL-6 IL-8 IFN-g TGF-b4 MHC-I
2.3. RNA isolation and quantitative real-time PCR Total RNA was extracted from chicken bursas by total RNA Kit (Omega Bio-Tek, Guangzhou, China) and then reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, CA, USA)
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MDA5 Actin
e indicates no reference.
e e e e e e e [18] [19] e
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4. Results
expressions were earlier than T cells infiltration.
4.1. Chemokine receptors gene expressions were earlier than T cells infiltration
4.2. CCR5 and CXCR4 antagonist treatments increased IBDV VP2 mRNA levels in bursa
The relationship between T cell infiltration into the bursae and chemokine receptors gene expression was displayed in Fig. 1. CD3þ T cells began to largely home into the bursae on Day 3 after infection with IBDV and increased to 29.4%. However, the mRNA of chemokine receptors, including CCR5 and CXCR4, in the surface of T cells began to increase only on Day 1 and then largely decreased to 5.1 and 0.13% on Day 3. In all, chemokine receptors gene
Post infection with IBDV, some clinical symptoms, such as decreased feed intake, fluffed feathers and diarrhea, were seen in chickens from the infected group. Pathological changes displayed that the bursa of Fabricius was hemorrhage and necrosis. Moreover, chickens from the Maraviroc-treated group and the AMD3100treated group demonstrated similar clinical symptoms with the infected group; some symptoms were even more serious. To
Fig. 1. Relationships between T cell infiltration and relative mRNA levels of CCR5 and CXCR4 in bursae by real-time PCR (n ¼ 3). (A) Total T cell percentage in burase; (B) Relative mRNA levels (mean ± SD) of chemokine receptor CCR5 in the bursa of Fabricius; (C) Relative mRNA levels (mean ± SD) of chemokine receptor CXCR4 in the bursa of Fabricius. ** Indicates P < 0.01 the infection group vs the mock group on Day 1 or Day 3 post-inoculation with IBDV.
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Fig. 2. Quantification of the infectious bursal disease virus (IBDV) VP2 gene levels the bursa of Fabricius in the experimental groups [the mock, infection, Maraviroc and AMD3100 group] using real-time PCR. The data were expressed as the average ratio of IBDV/b-actin. **Indicates P < 0.01 the infection group vs the chemokine receptor antagonist-treatment groups.
determine the effects of chemokine receptors antagonists on IBDV replication in bursa, IBDV VP2 genes in the bursa from the IBDVinfected group were quantified by real-time PCR on day 3 postinfection (Fig. 2). The results showed that VP2 gene expressions of the chemokine receptors antagonists-treated groups were two to three times higher than that of the infected group.
significantly higher than that of the mock group. Moreover, the CCR5 gene expressions of the Maraviroc-treated group and the AMD3100-treated group were approximately 5-fold and 3-fold higher than the CCR5 mRNA level of the infection group, respectively. However, IBDV infection induced the lower CXCR4 mRNA levels of the infection group than the mock group while the CXCR4 gene levels of the Maraviroc-treated group and the AMD3100treated group almost returned to the normal level.
4.3. Antagonist treatments did not decrease chemokine receptors CCR5 and CXCR4 mRNA levels of in bursa The mRNA contents of CCR5 and CXCR4 of bursa on day 3 post infection were quantified by real-time PCR and displayed different expression trends (Fig. 3). After inoculation with IBDV, the CCR5 mRNA levels of the bursa from the IBDV-infected groups were
4.4. Antagonist treatments influenced related cytokines mRNA expressions in bursa To explore why antagonist treatments increase IBDV VP2 gene levels, some related cytokines gene expressions were shown in
Fig. 3. Relative mRNA levels (mean þ SD) of chemokine receptors (A) CCR5 and (B) CXCR4 in the bursa of Fabricius (n ¼ 3). The mRNA levels were determined by quantitative realtime PCR analysis and were normalized to b-actin. **Indicates P < 0.01 when the infection group was compared with the chemokine receptor antagonist-treatment groups. # Indicates P < 0.05 when the AMD3100 group was compared with the Maraviroc group. ##Indicates P < 0.01 when the AMD3100 group was compared with the Maraviroc group.
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Fig. 4. The effect of Maraviroc and AMD3100 treatments on the levels of IL-2 (A), IFN-g (B), IL-6 (C), IL-8 (D), TGF-b4 (E), MHC-I (F) and MDA5 (G) in the bursa of Fabricius by realtime PCR on day 3 post infection with IBDV. **Indicates P < 0.01 when the infection group was compared with the chemokine receptor antagonist-treatment groups. ##Indicates P < 0.01 when the AMD3100 group was compared with the Maraviroc group. “c”Indicates P < 0.01 when the Maraviroc group was compared with the infection and AMD3100 groups.
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Fig. 4. Maraviroc and AMD3100 could significantly induce higher gene transcriptions of IL-2, IL-6, TGF-b4 and MHC-I compared with the infection group (P < 0.01). Moreover, the effects of Maraviroc on cytokines regulation, such as IL-2, IL-6, TGF-b4, MHC-I and MDA5, were better than AMD3100 did (P < 0.01). However, antagonist treatments did not influence the gene levels of IFN-g during IBDV infection, or even worse for IL-8 levels. 5. Discussions Normal and healthy chickens almost have no resident bursal T cell, however, lots of T cells infiltrated into the bursae accompanied with IBDV replication at the site of virus replication [21]. Influx of T cells modulated IBDV pathogenesis by limiting viral replication, promoting tissue damage and delaying tissue recovery [22]. However, the key factors which influenced T cell infiltration have not been known. Chemokine receptors played an important role in migrating T cells into different organs during health and disease development. CCR5 and CXCR4 have been reported to take part in migration, development and distribution of T cells and the present study has also demonstrated that both CCR5 and CXCR4 took part in the process of T cell migration during IBDV infection. Feeding CCR5 and CXCR4 antagonists could increase IBDV VP2 gene expression level and regulate cytokines expression levels induced by T cells. T cells have been shown to limit viral replication in the bursa in the early phase of IBDV infection; therefore, in other words, we speculated that T cell migration delay could increase virus replication in the early phase. Maraviroc is a CCR5 chemokine receptor antagonist while AMD3100 is used to be a highly selective CXCR4 receptor antagonist and potently inhibit SDF-1 binding [23e25]. Our study has demonstrated these antagonists have promoted virus replication on day 3 post inoculation with IBDV, of which the results verified our speculation. Actually, CCR5 has already been reported to be a host factor for Dengue virus-2 replication and disease development and CCR5 antagonist treatment led to a decrease in Dengue virus-2 titres throughout all time-points [17]. Moreover, immune cell recruitment into liver of HBx-TG mice was blocked by a CXCR4 antagonist [26]. These results indicated that blockade of some important chemokine receptor exerted substantial role on virus replication and disease development. Influx of T cells into the bursa induced high expressions levels of CCR5 and low gene content of CXCR4 in the bursa in the present study, which was very interesting. Both CCR5 and CXCR4 were the important chemokine receptor expressed on the surface of T cells, why did they display the different gene expression levels? Moreover, feeding antagonists could increase CCR5 gene levels and offset a downward trend of CXCR4 gene levels in bursa from chickens inoculated with IBDV. We believed that excessive T cells migration into bursa induced high CCR5 gene expressions, though the T cells could not inhibit IBDV reproduction after chemokine receptor antagonist administration. Meanwhile, CXCR4 gene levels only returned to the normal level after chemokine receptor antagonist treatment due to the possibility of inhibiting CXCR4 gene expressions induced by IBDV infection. The CCR5 expression was severalfold increase in the inflammatory cerebrospinal fluid in the process of flavivirus encephalitis, but CCR5 expression decreased in the CCR5D32/wt genotype patients without a compensatory increase of its ligand CCL5 expression, indicating CCR5 expression in the activated lymphocyte population is influenced during flavivirus encephalitis [27]. Previous study has demonstrated that the CXCL12/ CXCR4 pathway exerted huge effects in human papillomavirusinduced pathogenesis and selectively blockade of this axis was a potentially treatment option, of which the results were inconsistent with our results [28]. Therefore, during the progress of IBDV infection and T cell migration, CCR5 and CXCR4 played a different
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role and further study should be focused on the mechanisms of their different functions. In order to explain why virus replication was strengthened after antagonist treatment, some cytokines and immune-related genes were determined by qRT-PCR. Gene transcription of proinflammatory cytokines IL-6 and IL-8 exhibits up-regulation during IBDV infection [29]. However, the present study revealed that antagonist treatments could elevate IL-6 gene levels and decrease IL-8 gene level compared with the infection group. Th1 cytokines IL-2 and IFN-g was also significantly increased to activate T cells in the process of IBDV infection, similarly, administration with Maraviroc and AMD3100 also induced higher levels of Th1 cytokines than the infection group [30]. Anti-inflammatory cytokine TGF-b4, antiviral activation genes MHC-I and MDA5 as one member of RIGI-like receptors were all promoted gene expressions after antagonist treatment compared with the infection group [19,29]. Chemokine receptor antagonists could elevate gene transcriptions of these pro-inflammatory cytokine, Th1 cytokines, antiinflammatory cytokine and other related genes except IL-8 compared with the infection group; meanwhile, the effects of Maraviroc seemed to be more effective than AMD3100 did, which might explain why the virus loads in the Maraviroc-treated group were higher than those of the AMD3100-treated group. Author's contribution OC and WQ designed the experiment and drafted the manuscript. YZ and YY participated in the experiment and bioinformatics analysis of the sequencing data. YY and MJ performed the molecular-related experiments. LX and KX participated in the design of the experiment, the discussion and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (No.31402169 and 31602024), China Postdoctoral Science Foundation funded project (No.2015M570630), Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 13HASTIT006), Key scientific research projects of Henan Province (No.15A230002) and post doctoral fund of Henan institute of science and technology. References [1] T. Ruby, C. Whittaker, D.R. Withers, M.K. Chelbi-Alix, V. Morin, A. Oudin, J.R. Young, R. Zoorob, Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection, J. Virology 80 (18) (2006) 9207e9216. [2] F. Rauw, B. Lambrecht, T. van den Berg, Pivotal role of ChIFNgamma in the pathogenesis and immunosuppression of infectious bursal disease, Avian Pathology J. WVPA 36 (5) (2007) 367e374. [3] B. Homey, A. Muller, A. Zlotnik, Chemokines: agents for the immunotherapy of cancer? Nat. Rev. Immunol. 2 (3) (2002) 175e184. [4] S. Serrano-Villar, T. Sainz, Z.M. Ma, N.S. Utay, T. Wook-Chun, S. Mann, A.D. Kashuba, B. Siewe, A. Albanese, P. Troia-Cancio, et al., Effects of combined CCR5/integrase inhibitors-based regimen on mucosal immunity in HIVinfected patients naive to antiretroviral therapy: a pilot randomized trial, PLoS Pathog. 12 (1) (2016) e1005381. [5] B. Debnath, S. Xu, F. Grande, A. Garofalo, N. Neamati, Small molecule inhibitors of CXCR4, Theranostics 3 (1) (2013) 47e75. [6] S. Oishi, N. Fujii, Peptide and peptidomimetic ligands for CXC chemokine receptor 4 (CXCR4), Org. Biomol. Chem. 10 (30) (2012) 5720e5731. [7] G. Fatkenheuer, A.L. Pozniak, M.A. Johnson, A. Plettenberg, S. Staszewski, A.I. Hoepelman, M.S. Saag, F.D. Goebel, J.K. Rockstroh, B.J. Dezube, et al., Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1, Nat. Med. 11 (11) (2005) 1170e1172. [8] C.A. Toupadakis, A. Wong, D.C. Genetos, D.J. Chung, D. Murugesh, M.J. Anderson, G.G. Loots, B.A. Christiansen, A.S. Kapatkin, C.E. Yellowley, Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair, J. Orthop. Res. Official Publ. Orthop. Res. Soc.
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