Veterinary Immunology and Immunopathology 164 (2015) 51–55
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Chicken bone marrow-derived dendritic cells maturation in response to infectious bursal disease virus Jinfeng Liang, Yinyan Yin, Tao Qin, Qian Yang ∗ Key Lab of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Weigang No. 1, Nanjing 210095, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 23 March 2014 Received in revised form 11 December 2014 Accepted 31 December 2014 Keywords: Bone marrow Chicken Dendritic cells Infectious bursal disease virus
a b s t r a c t Infectious bursal disease virus (IBDV) is highly contagious disease which easily lead to immunosuppression and a decreased response to vaccinations in young chicken. Since dendritic cells (DCs) are crucial to induce immunity and their maturation and functions are influenced by microbial and environmental stimuli, we investigated the effects of inactivated IBDV and IBDV on chicken DC activation and maturation. Chicken bone marrowderived dendritic cells (chBM-DCs) cultured in complete medium (including recombinant chicken: granulocyte-macrophage colony-stimulating factor and interleukin 4) expressed high levels of MHC-II and the putative CD11c. After LPS or virus stimulation, chBM-DCs displayed the typical morphology of DCs. In addition, stimulation by LPS or viruses significantly elevated chBM-DCs surface expression levels of CD40 and CD86 molecules, as well as the ability to induce T-cell proliferative response, compared to the non-stimulated chBM-DCs. Interestingly, inactive IBDV showed stronger ability to up-regulate expression levels of CD40 and CD86 molecules and stimulate naive T cells proliferation than live IBDV. These results revealed that live viruses infection impaired DC maturation and functions, probably explaining why chickens infected with IBDV fails to trigger an effective specific immune response or develop immune memory. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Infectious bursal disease virus (IBDV), a member of the Birnaviridae family whose genome consists of two segments of double stranded RNA, causes an acute, highly contagious disease in young chickens (Jacobs and Damania, 2012; Rautenschlein et al., 2002). The disease has an important economic impact on the poultry industry worldwide because it is associated with a high mortality and immunosuppression in recovered chickens, which leads both to a variety of secondary infections and a decreased response
∗ Corresponding author. Tel.: +86 02584395817; fax: +86 02584398669. E-mail address: [email protected] (Q. Yang). http://dx.doi.org/10.1016/j.vetimm.2014.12.012 0165-2427/© 2015 Elsevier B.V. All rights reserved.
to vaccinations (Roh et al., 2006). Immune responses are readily measurable, which are effective for the control of IBDV in chickens. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique ability to induce both innate immune responses and a highly specific acquired immunity (Banchereau and Steinman, 1998). Avian DCs were first reported in the cecal tonsils, the secondary lymphoid organs located in the intestinal mucosa (Olah and Glick, 1979). Since then, three major subsets of DCs (inter-digitating DCs, follicular DCs, and epidermal DCs) also were identified in chicken lymphoid tissues or epidermis (Del Cacho et al., 2009; Igyártó et al., 2006). Immature DCs become mature when they sense pathogen-associated molecular patterns released from pathogens through a limited number of pattern recognition receptors (PRRs). Several classes of PRRs expressed on
52
J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
DCs, including Toll-like receptors (TLRs) and cytoplasmic receptors, can recognize distinct microbial components and directly initiate signal pathways, ultimately resulting in the activation of immunity (Akira et al., 2006). In recent years, with the emergence of new chicken DCs markers, it has become increasingly possible to culture chicken DCs in vitro (Fu et al., 2014; Wu et al., 2010). There is currently no knowledge about the nature of virus interactions with chicken bone marrow-derived dendritic cells (chBM-DCs). Therefore, in this study we decided to investigate the activation and maturation of chBM-DCs by stimulation with IBDV and inactivated-IBDV in an attempt to understand the involvement of chBMDCs in the immune suppressive diseases caused by IBDV infection.
2. Materials and methods 2.1. Chickens and viruses Specific pathogen-free (SPF) ROSS 308 chickens (4–6week-old) were obtained from Jiangsu Academy of Agricultural Sciences (Nanjing, China), and maintained at an animal facility under pathogen free conditions. A tissue culture infectious dose 50 of 10−6.38 /0.1 ml of the IBDV strain (B87; Jiangsu Academy of Agricultural Sciences, China) was used. Virus was heat-inactivated at 56 ◦ C for 30 min.
2.2. Culture of chBM-DCs ChBM-DCs were generated from 4 to 6-week-old inbred line ROSS 308 chickens as previously described (Wu et al., 2010; Liang et al., 2013). Briefly, Chicken bone marrow precursors were obtained from femurs and tibias and cultured at a final concentration of 2 × 106 cells/ml in six-well plates in 3 ml the culture medium containing RPMI-1640 (GIBICO, USA), 10% fetal bovine serum (FBS) (Wisent, CAN), 50 ng/ml recombinant chicken GM-CSF (Abcam, USA) (ab119158), 10 ng/ml recombin-ant chicken IL-4 (Kingfisher, USA) (RP0110C-025), 1 U/ml penicillin and 1 g/ml streptomycin, for 7 days at 37 ◦ C, 5% CO2 . Half of the medium was replaced with fresh, prewarmed complete medium at day 2 and day 4 to remove non-adherent cells (such as dead cells and granulocytes). Immature chBMDC aggregates started to grow from day 4. On day 6, LPS (200 ng/ml, Sigma-Aldrich) IBDV (10 l/ml) or inactivatedIBDV (10 l/ml) was used to stimulate immature chBM-DC for 24 h. At day 7 of culture, all cells were harvested by gentle pipetting and centrifugal separation.
2.4. Flow cytometry analysis The harvested cells were washed one with 0.01 M phosphate buffered saline (PBS). The un-stimulated chBM-DCs (0.5 × 106 cells/ml) were incubated with 0.05 mg/ml of PE-conjugated mouse anti-human CD11c antibody (eBioscience, USA) (Fu et al., 2014) or 0.5 mg/ml of Fluorescein isothiocyanate (FITC)-labeled mouse anti-chicken MHC-II antibody (Abcam, USA) for 20 min at 25 ◦ C. In addition, the stimulated chBM-DCs were incubated with 25 g/ml of mouse anti-chicken CD40 or CD86 antibody (Abd, UK) for 20 min at 25 ◦ C. After being washed, the cells were then stained with PE-conjugated Goat anti-mouse IgG second antibody diluted 1:5000 (MultiScience, China) for 15 min at 25 ◦ C. After the final wash with PBS, the levels of fluorescence from DCs were determined by a FACSCalibur (BD Bioscience, Cowley, UK). 2.5. T-cell proliferation assays To determine the T-cell stimulatory capacity of chBMDCs, allogeneic mixed lymphocyte reaction (MLR) of DCs was assayed as previously described (Wu and Kaiser, 2011). Briefly, after cultured with the complete medium for 6 days, the DCs were stimulated with viruse or LPS, respectively, for 24 h before allogeneic MLR. Allogeneic T lymphocytes were isolated from spleen of 4–6 week-old chickens and purified on nylon wood column. T lymphocytes were added to 96-well round-bottomed cell culture plates and 1 × 104 DCs were added, giving a ration of T cells: DCs of 1:1, 10:1, 100:1, in a culture volume of 100 l. Control cultures contained T cells or DCs cells only. All experiments were performed at least in triplicate. The cells were cultured for two additional days in 5% CO2 at 37 ◦ C, and then T cells proliferation was determined by cell counting kit8 (CCK-8) assay (Beyotime, Jiangsu, China) according to the manufacture’s instruction. Cells in 96-well plate were added with 10 l CCK-8 solution, and incubated for 2 h at 37 ◦ C. Absorbances of each well were read at 450 nm using an automated ELISA reader (Bio-Tech instruments, USA). The Stimulation Index was calculated as formula: SI = (ODsample − ODDCs only )/(ODT cells only − ODblank control ). 2.6. Statistical analysis All date analysis was performed one-way ANOVA analysis in SPSS 17.0. Differences were considered statistically significant at p < 0.05. 3. Results
2.3. Observation of morphology
3.1. The morphologies of non-stimulated cells and viruses-stimulated cells when cultured in the presence of GM-CSF and IL-4
Effects of recombinant chicken GM-CSF and IL-4 on cell differentiation were recorded by observing cell morphology, clustering and cell growth at day 4 and day 7. The stimulatory effects of LPS and IBDV on cells maturation were observed with a digital camera on an inverted microscope on day 7.
Cells were cultured in the presence of GM-CSF and IL4 for 4 day; many cell aggregates were seen under an inverted light microscope (Fig. 1A). After viruses stimulation on day 6, these cells aggregates grew and became bigger and floating or loosely adherent (Fig. 1B). In addition, cells stimulated by viruses showed dendrites (Fig. 1C)
J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
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Fig. 1. Morphologies of non-stimulated cells and viruses-stimulated cells: (A) Cell aggregates were observed at day 4 with a light microscope; (B) cell aggregates increased after stimulation with viruses for 24 h; (C) single cells showed dendrites (arrow) after stimulation with viruses for 24 h; (D) culture of chicken bone marrow-derived dendritic cells at day 7 in the absence of GM-CSF and IL-4. (E) Culture of chicken bone marrow-derived dendritic cells at day 7 in the presence of recombinant chicken GM-CSF alone. (F) Culture of chicken bone marrow-derived dendritic cells at day 7 in the presence of recombinant chicken IL-4 alone.
that is considered as a sign of maturation (Tao, 1998). When bone marrow precursors were cultured in the absence of GM-CSF and IL-4, no cell aggregates were observed and only a few live cells were left in the plates by day 7 (Fig. 1D). When cells were cultivated with GM-CSF alone, aggregates were also seen on day 7(Fig. 1E). However, when cells were cultivated with IL-4 alone, no aggregates were observed on day 7 (Fig. 1F).
to evaluate the purity of DCs (Fu et al., 2014; Wu et al., 2010). The immature DCs expressed high levels of cell surface MHC II and putative CD11c molecules (Fig. 2A). When LPS, inactivated IBDV or live IBDV were used to stimulate the DCs, there were significantly increased expressions of CD40 and CD86 on DCs (Fig. 2B). LPS stimulated 200% more and inactivated IBDV 103% more when compared to live IBDV.
3.2. Non-stimulated cells had the phenotype of immature DCs and viruses-stimulated cells had the phenotype of mature DCs
3.3. Viruses-stimulated chBM-DCs stimulated proliferation of naïve T cells in MLR
The phenotypic analysis of chBM-DCs by flow cytometry was shown in Fig. 2. In birds, the expression levels of MHC II and putative CD11c on immature DCs were used
Non-stimulated chBM-DCs stimulated poor allogeneic cells in MLR, compared with controls of T cells only. However, the chBM-DCs stimulated by LPS, IBDV and inactivated-IBDV efficiently evoked allostimulatory
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J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
Fig. 2. Comparative flow cytometric analysis of cell surface molecule expression on chicken bone marrow-derived dendritic cells after stimulation with viruses: (A) Immature chicken dendritic cells showed high expression of putative CD11c and MHC Class II+. The black lines display staining with the indicated specific antibody and the grey lines represented the isotype control. (B) A significant increase of cell surface CD40 and CD86 was observed on both LPS-stimulated and viruses-stimulated cells, compared by non-stimulated chicken dendritic cells. The black lines display staining with anti-chicken cell surface marker monoclonal antibodies and the grey lines represented the isotype control. The results shown were representative of three independent experiments.
4. Discussion
Fig. 3. Viruses-stimulated chicken bone marrow-derived dendritic cells stimulated proliferation of naive T cells in allogeneic MLR: Responder cells were isolated from spleen. Stimulator cells were chicken dendritic cells stimulated with LPS, IBDV, inactivated IBDV or not stimulated at 37 ◦ C for 24 h. Control cultures contained responder or stimulator cells only. All experiments were performed at least in triplicates. Significant differences between the treated groups and the control groups were expressed as * p ≤ 0.05 and ** p ≤ 0.01.
capacity in MLR (p < 0.01 or p < 0.05), compared to nonstimulated chBM-DCs (Fig. 3). In addition, inactivated-IBDV was more efficient in inducing T cells proliferation at a 1:1 ratio of allogeneic T cells and chBM-DCs when compared to IBDV.
DCs perform a sentinel function for the recognition of invading pathogens and a regulatory function to control both innate and adaptive immunity (Levitz and Golenbock, 2012). Immature DCs capture and process antigen, become mature with up regulation of co-stimulatory molecules such as MHC II, CD40, CD80 and CD86. Beside, only the mature DCs can stimulate T cells efficiently (Tan and O’Neill, 2005). Numeral studies have established that DCs maturate in response to various viruses in mammals (Brimnes et al., 2003; López et al., 2004). However it does not appear to investigate the different effects of inactivated IBDV and IBDV on DC activation and maturation in birds. In this study, we observed an ability of DCs to express high levels of the maturation markers CD86 and CD40 when exposed to the inactivated-IBDV, suggesting that immature chBM-DCs could recognize conserved structural moieties of inactivated-IBDV and become phenotypically mature. This might be attributed to the activation of TLR signal pathways by binding of viruses to their corresponded TLR on chBM-DCs. TLR3 might be largely engaged in this process, which can recognize viral double-stranded RNA in birds (Chen et al., 2013). However, we also observed that chBM-DCs expressed low levels of the maturation markers CD86 and CD40 in response to IBDV. We inferred that the direct infection of DCs by IBDV may trigger a viral scape mechanism to
J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
impair DC function and impede the initiation of an effective immune response. Previous researchers reported that IAV-infected human myeloid DCs were impaired in their ability to cross-present exogenous antigen via MHCI and further inhibit DCs maturation (Waithman and Mintern, 2012). Ruth Chin et al. also have previously shown that the different viral strains infection could produce different proteins and RNA within human DCs. Such virus strain differenced in infection of human DCs could account for the different capacities to stimulate DCs maturation (Chin et al., 2013). In this regard, we deduce that the IBDV strain infection specifically inhibited chBM-DCs maturation. It is well known that only DCs are capable of activating naïve T cells and mature DCs have a strong capacity to induce proliferation of allogeneic T cell (Harris and Fabry, 2012). We therefore characterized their ability to drive strong allogeneic MLR when mature. In our studies, inactivated-IBDV or IBDV stimulated-DCs could dramatically evoked the ability to stimulate proliferation of naive T cells in the allogeneic MLR, suggesting that the chBMDCs took up and processed these viruses for presentation to T cells in vitro (Nierkens et al., 2013). Compared to IBDV, inactivated-IBDV had better effect on stimulating primary T cell responses and this might be correlated well with the higher expression of the co-stimulatory molecules CD40 and CD86 on inactivated-IBDV stimulated-DCs. Another reason for the lower T-cell proliferation of IBDV-stimulated DC is that the altered response was dependent on the viral protein 2 and did not require infection of DC with IBDV (Azizi et al., 2013). Other reasons for the dose-dependent proliferative response may be that properties of DC that contribute to successful T-cell activation (Chen et al., 2013). In conclusion, we successfully cultured chBM-DCs which had been characterized in terms of their morphology and high purity in vitro. We demonstrated that inactivated-IBDV in contrast to live IBDV was better to induce phenotypical and functional maturation of chBMDCs. This is the first report that inactivated IBDV is strong inducers of chBM-DCs maturation. These results suggest a novel mechanism to explain why infection of live IBDV fails to trigger an effective specific immune response or develop immune memory. Exploring the mechanisms involved in enhancement of the immune response by IBDV may greatly accelerate vaccine development. Conflict of interest statement There were no potential conflicts (financial, professional or personal) that are relevant to the manuscript. Acknowledgments This work was supported by the National Science Grant of P. R. China (No. 31172302) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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References Akira, S., Uematsu, S., Takeuchi, O., 2006. Pathogen recognition and innate immunity. Cell 124, 783–801. Azizi, M., Yakhchali, B., Ghamarian, A., Enayati, S., Khodabandeh, M., Khalaj, V., 2013. Cloning and expression of gumboro VP2 antigen in Aspergillus niger. Avicenna J. Med. Biotechnol. 5, 35. Banchereau, J., Steinman, R.M., 1998. Dendritic cells and the control of immunity. Nature 392, 245–252. Brimnes, M.K., Bonifaz, L., Steinman, R.M., Moran, T.M., 2003. Influenza virus-induced dendritic cell maturation is associated with the induction of strong T cell immunity to a coadministered, normally nonimmunogenic protein. J. Exp. Med. 198, 133–144. Chen, S., Cheng, A., Wang, M., 2013. Innate sensing of viruses by pattern recognition receptors in birds. Vet. Res. 44, 82. Chin, R., Earnest-Silviera, L., Gordon, C.L., Olsen, K., Barr, I., Brown, L.E., Jackson, D.C., Torresi, J., 2013. Impaired dendritic cell maturation in response to pandemic H1N109 influenza virus. J. Clin. Virol. 56, 226–231 (the official publication of the Pan American Society for Clinical Virology). Del Cacho, E., Gallego, M., Lillehoj, H.S., Lopez-Bernard, F., Sanchez-Acedo, C., 2009. Avian follicular and interdigitating dendritic cells: isolation and morphologic, phenotypic, and functional analyses. Vet. Immunol. Immunopathol. 129, 66–75. Fu, J., Liang, J., Kang, H., Lin, J., Yu, Q., Yang, Q., 2014. The stimulatory effect of different CpG oligonucleotides on the maturation of chicken bone marrow-derived dendritic cells. Poult. Sci. 93, 63–69. Harris, M.G., Fabry, Z., 2012. Initiation and regulation of CNS autoimmunity: balancing immune surveillance and inflammation in the CNS. Neurosci. Med. 3, 203–224. Igyártó, B.Z., Lackó, E., Oláh, I., Magyar, A., 2006. Characterization of chicken epidermal dendritic cells. Immunology 119, 278–288. Jacobs, S.R., Damania, B., 2012. NLRs, inflammasomes, and viral infection. J. Leukoc. Biol. 92, 469–477. Levitz, S.M., Golenbock, D.T., 2012. Beyond empiricism: informing vaccine development through innate immunity research. Cell 148, 1284–1292. Liang, J., Fu, J., Kang, H., Lin, J., Yu, Q., Yang, Q., 2013. The stimulatory effect of TLRs ligands on maturation of chicken bone marrow-derived dendritic cells. Vet. Immunol. Immunopathol. 155, 205–210. López, C.B., Moltedo, B., Alexopoulou, L., Bonifaz, L., Flavell, R.A., Moran, T.M., 2004. TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses. J. Immunol. 173, 6882–6889. Nierkens, S., Tel, J., Janssen, E., Adema, G.J., 2013. Antigen crosspresentation by dendritic cell subsets: one general or all sergeants? Trends Immunol. 34, 361–370. Olah, I., Glick, B., 1979. Structure of the germinal centers in the chicken caecal tonsil: light and electron microscopic and autoradiographic studies. Poult. Sci. 58, 195–210. Rautenschlein, S., Yeh, H.-Y., Sharma, J., 2002. The role of T cells in protection by an inactivated infectious bursal disease virus vaccine. Vet. Immunol. Immunopathol. 89, 159–167. Roh, H.J., Sung, H.W., Kwon, H.M., 2006. Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-␥on protective immunity by a DNA vaccine against IBDV in chickens. J. Vet. Sci. 7, 361–368. Tan, J.K., O’Neill, H.C., 2005. Maturation requirements for dendritic cells in T cell stimulation leading to tolerance versus immunity. J. Leukoc. Biol. 78, 319–324. Tao, W., 1998. Dendritic cells and anti-infection immunity. Int. J. Immunol. 21, 311–314. Waithman, J., Mintern, J.D., 2012. Dendritic cells and influenza A virus infection. Virulence 3, 603–608. Wu, Z., Kaiser, P., 2011. Antigen presenting cells in a non-mammalian model system, the chicken. Immunobiology 216, 1177– 1183. Wu, Z., Rothwell, L., Young, J.R., Kaufman, J., Butter, C., Kaiser, P., 2010. Generation and characterization of chicken bone marrow-derived dendritic cells. Immunology 129, 133–145.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Chicken bone marrow-derived dendritic cells maturation in response to infectious bursal disease virus Jinfeng Liang, Yinyan Yin, Tao Qin, Qian Yang ∗ Key Lab of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Weigang No. 1, Nanjing 210095, Jiangsu, China
a r t i c l e
i n f o
Article history: Received 23 March 2014 Received in revised form 11 December 2014 Accepted 31 December 2014 Keywords: Bone marrow Chicken Dendritic cells Infectious bursal disease virus
a b s t r a c t Infectious bursal disease virus (IBDV) is highly contagious disease which easily lead to immunosuppression and a decreased response to vaccinations in young chicken. Since dendritic cells (DCs) are crucial to induce immunity and their maturation and functions are influenced by microbial and environmental stimuli, we investigated the effects of inactivated IBDV and IBDV on chicken DC activation and maturation. Chicken bone marrowderived dendritic cells (chBM-DCs) cultured in complete medium (including recombinant chicken: granulocyte-macrophage colony-stimulating factor and interleukin 4) expressed high levels of MHC-II and the putative CD11c. After LPS or virus stimulation, chBM-DCs displayed the typical morphology of DCs. In addition, stimulation by LPS or viruses significantly elevated chBM-DCs surface expression levels of CD40 and CD86 molecules, as well as the ability to induce T-cell proliferative response, compared to the non-stimulated chBM-DCs. Interestingly, inactive IBDV showed stronger ability to up-regulate expression levels of CD40 and CD86 molecules and stimulate naive T cells proliferation than live IBDV. These results revealed that live viruses infection impaired DC maturation and functions, probably explaining why chickens infected with IBDV fails to trigger an effective specific immune response or develop immune memory. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Infectious bursal disease virus (IBDV), a member of the Birnaviridae family whose genome consists of two segments of double stranded RNA, causes an acute, highly contagious disease in young chickens (Jacobs and Damania, 2012; Rautenschlein et al., 2002). The disease has an important economic impact on the poultry industry worldwide because it is associated with a high mortality and immunosuppression in recovered chickens, which leads both to a variety of secondary infections and a decreased response
∗ Corresponding author. Tel.: +86 02584395817; fax: +86 02584398669. E-mail address: [email protected] (Q. Yang). http://dx.doi.org/10.1016/j.vetimm.2014.12.012 0165-2427/© 2015 Elsevier B.V. All rights reserved.
to vaccinations (Roh et al., 2006). Immune responses are readily measurable, which are effective for the control of IBDV in chickens. Dendritic cells (DCs) are professional antigen-presenting cells (APCs) with the unique ability to induce both innate immune responses and a highly specific acquired immunity (Banchereau and Steinman, 1998). Avian DCs were first reported in the cecal tonsils, the secondary lymphoid organs located in the intestinal mucosa (Olah and Glick, 1979). Since then, three major subsets of DCs (inter-digitating DCs, follicular DCs, and epidermal DCs) also were identified in chicken lymphoid tissues or epidermis (Del Cacho et al., 2009; Igyártó et al., 2006). Immature DCs become mature when they sense pathogen-associated molecular patterns released from pathogens through a limited number of pattern recognition receptors (PRRs). Several classes of PRRs expressed on
52
J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
DCs, including Toll-like receptors (TLRs) and cytoplasmic receptors, can recognize distinct microbial components and directly initiate signal pathways, ultimately resulting in the activation of immunity (Akira et al., 2006). In recent years, with the emergence of new chicken DCs markers, it has become increasingly possible to culture chicken DCs in vitro (Fu et al., 2014; Wu et al., 2010). There is currently no knowledge about the nature of virus interactions with chicken bone marrow-derived dendritic cells (chBM-DCs). Therefore, in this study we decided to investigate the activation and maturation of chBM-DCs by stimulation with IBDV and inactivated-IBDV in an attempt to understand the involvement of chBMDCs in the immune suppressive diseases caused by IBDV infection.
2. Materials and methods 2.1. Chickens and viruses Specific pathogen-free (SPF) ROSS 308 chickens (4–6week-old) were obtained from Jiangsu Academy of Agricultural Sciences (Nanjing, China), and maintained at an animal facility under pathogen free conditions. A tissue culture infectious dose 50 of 10−6.38 /0.1 ml of the IBDV strain (B87; Jiangsu Academy of Agricultural Sciences, China) was used. Virus was heat-inactivated at 56 ◦ C for 30 min.
2.2. Culture of chBM-DCs ChBM-DCs were generated from 4 to 6-week-old inbred line ROSS 308 chickens as previously described (Wu et al., 2010; Liang et al., 2013). Briefly, Chicken bone marrow precursors were obtained from femurs and tibias and cultured at a final concentration of 2 × 106 cells/ml in six-well plates in 3 ml the culture medium containing RPMI-1640 (GIBICO, USA), 10% fetal bovine serum (FBS) (Wisent, CAN), 50 ng/ml recombinant chicken GM-CSF (Abcam, USA) (ab119158), 10 ng/ml recombin-ant chicken IL-4 (Kingfisher, USA) (RP0110C-025), 1 U/ml penicillin and 1 g/ml streptomycin, for 7 days at 37 ◦ C, 5% CO2 . Half of the medium was replaced with fresh, prewarmed complete medium at day 2 and day 4 to remove non-adherent cells (such as dead cells and granulocytes). Immature chBMDC aggregates started to grow from day 4. On day 6, LPS (200 ng/ml, Sigma-Aldrich) IBDV (10 l/ml) or inactivatedIBDV (10 l/ml) was used to stimulate immature chBM-DC for 24 h. At day 7 of culture, all cells were harvested by gentle pipetting and centrifugal separation.
2.4. Flow cytometry analysis The harvested cells were washed one with 0.01 M phosphate buffered saline (PBS). The un-stimulated chBM-DCs (0.5 × 106 cells/ml) were incubated with 0.05 mg/ml of PE-conjugated mouse anti-human CD11c antibody (eBioscience, USA) (Fu et al., 2014) or 0.5 mg/ml of Fluorescein isothiocyanate (FITC)-labeled mouse anti-chicken MHC-II antibody (Abcam, USA) for 20 min at 25 ◦ C. In addition, the stimulated chBM-DCs were incubated with 25 g/ml of mouse anti-chicken CD40 or CD86 antibody (Abd, UK) for 20 min at 25 ◦ C. After being washed, the cells were then stained with PE-conjugated Goat anti-mouse IgG second antibody diluted 1:5000 (MultiScience, China) for 15 min at 25 ◦ C. After the final wash with PBS, the levels of fluorescence from DCs were determined by a FACSCalibur (BD Bioscience, Cowley, UK). 2.5. T-cell proliferation assays To determine the T-cell stimulatory capacity of chBMDCs, allogeneic mixed lymphocyte reaction (MLR) of DCs was assayed as previously described (Wu and Kaiser, 2011). Briefly, after cultured with the complete medium for 6 days, the DCs were stimulated with viruse or LPS, respectively, for 24 h before allogeneic MLR. Allogeneic T lymphocytes were isolated from spleen of 4–6 week-old chickens and purified on nylon wood column. T lymphocytes were added to 96-well round-bottomed cell culture plates and 1 × 104 DCs were added, giving a ration of T cells: DCs of 1:1, 10:1, 100:1, in a culture volume of 100 l. Control cultures contained T cells or DCs cells only. All experiments were performed at least in triplicate. The cells were cultured for two additional days in 5% CO2 at 37 ◦ C, and then T cells proliferation was determined by cell counting kit8 (CCK-8) assay (Beyotime, Jiangsu, China) according to the manufacture’s instruction. Cells in 96-well plate were added with 10 l CCK-8 solution, and incubated for 2 h at 37 ◦ C. Absorbances of each well were read at 450 nm using an automated ELISA reader (Bio-Tech instruments, USA). The Stimulation Index was calculated as formula: SI = (ODsample − ODDCs only )/(ODT cells only − ODblank control ). 2.6. Statistical analysis All date analysis was performed one-way ANOVA analysis in SPSS 17.0. Differences were considered statistically significant at p < 0.05. 3. Results
2.3. Observation of morphology
3.1. The morphologies of non-stimulated cells and viruses-stimulated cells when cultured in the presence of GM-CSF and IL-4
Effects of recombinant chicken GM-CSF and IL-4 on cell differentiation were recorded by observing cell morphology, clustering and cell growth at day 4 and day 7. The stimulatory effects of LPS and IBDV on cells maturation were observed with a digital camera on an inverted microscope on day 7.
Cells were cultured in the presence of GM-CSF and IL4 for 4 day; many cell aggregates were seen under an inverted light microscope (Fig. 1A). After viruses stimulation on day 6, these cells aggregates grew and became bigger and floating or loosely adherent (Fig. 1B). In addition, cells stimulated by viruses showed dendrites (Fig. 1C)
J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
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Fig. 1. Morphologies of non-stimulated cells and viruses-stimulated cells: (A) Cell aggregates were observed at day 4 with a light microscope; (B) cell aggregates increased after stimulation with viruses for 24 h; (C) single cells showed dendrites (arrow) after stimulation with viruses for 24 h; (D) culture of chicken bone marrow-derived dendritic cells at day 7 in the absence of GM-CSF and IL-4. (E) Culture of chicken bone marrow-derived dendritic cells at day 7 in the presence of recombinant chicken GM-CSF alone. (F) Culture of chicken bone marrow-derived dendritic cells at day 7 in the presence of recombinant chicken IL-4 alone.
that is considered as a sign of maturation (Tao, 1998). When bone marrow precursors were cultured in the absence of GM-CSF and IL-4, no cell aggregates were observed and only a few live cells were left in the plates by day 7 (Fig. 1D). When cells were cultivated with GM-CSF alone, aggregates were also seen on day 7(Fig. 1E). However, when cells were cultivated with IL-4 alone, no aggregates were observed on day 7 (Fig. 1F).
to evaluate the purity of DCs (Fu et al., 2014; Wu et al., 2010). The immature DCs expressed high levels of cell surface MHC II and putative CD11c molecules (Fig. 2A). When LPS, inactivated IBDV or live IBDV were used to stimulate the DCs, there were significantly increased expressions of CD40 and CD86 on DCs (Fig. 2B). LPS stimulated 200% more and inactivated IBDV 103% more when compared to live IBDV.
3.2. Non-stimulated cells had the phenotype of immature DCs and viruses-stimulated cells had the phenotype of mature DCs
3.3. Viruses-stimulated chBM-DCs stimulated proliferation of naïve T cells in MLR
The phenotypic analysis of chBM-DCs by flow cytometry was shown in Fig. 2. In birds, the expression levels of MHC II and putative CD11c on immature DCs were used
Non-stimulated chBM-DCs stimulated poor allogeneic cells in MLR, compared with controls of T cells only. However, the chBM-DCs stimulated by LPS, IBDV and inactivated-IBDV efficiently evoked allostimulatory
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J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
Fig. 2. Comparative flow cytometric analysis of cell surface molecule expression on chicken bone marrow-derived dendritic cells after stimulation with viruses: (A) Immature chicken dendritic cells showed high expression of putative CD11c and MHC Class II+. The black lines display staining with the indicated specific antibody and the grey lines represented the isotype control. (B) A significant increase of cell surface CD40 and CD86 was observed on both LPS-stimulated and viruses-stimulated cells, compared by non-stimulated chicken dendritic cells. The black lines display staining with anti-chicken cell surface marker monoclonal antibodies and the grey lines represented the isotype control. The results shown were representative of three independent experiments.
4. Discussion
Fig. 3. Viruses-stimulated chicken bone marrow-derived dendritic cells stimulated proliferation of naive T cells in allogeneic MLR: Responder cells were isolated from spleen. Stimulator cells were chicken dendritic cells stimulated with LPS, IBDV, inactivated IBDV or not stimulated at 37 ◦ C for 24 h. Control cultures contained responder or stimulator cells only. All experiments were performed at least in triplicates. Significant differences between the treated groups and the control groups were expressed as * p ≤ 0.05 and ** p ≤ 0.01.
capacity in MLR (p < 0.01 or p < 0.05), compared to nonstimulated chBM-DCs (Fig. 3). In addition, inactivated-IBDV was more efficient in inducing T cells proliferation at a 1:1 ratio of allogeneic T cells and chBM-DCs when compared to IBDV.
DCs perform a sentinel function for the recognition of invading pathogens and a regulatory function to control both innate and adaptive immunity (Levitz and Golenbock, 2012). Immature DCs capture and process antigen, become mature with up regulation of co-stimulatory molecules such as MHC II, CD40, CD80 and CD86. Beside, only the mature DCs can stimulate T cells efficiently (Tan and O’Neill, 2005). Numeral studies have established that DCs maturate in response to various viruses in mammals (Brimnes et al., 2003; López et al., 2004). However it does not appear to investigate the different effects of inactivated IBDV and IBDV on DC activation and maturation in birds. In this study, we observed an ability of DCs to express high levels of the maturation markers CD86 and CD40 when exposed to the inactivated-IBDV, suggesting that immature chBM-DCs could recognize conserved structural moieties of inactivated-IBDV and become phenotypically mature. This might be attributed to the activation of TLR signal pathways by binding of viruses to their corresponded TLR on chBM-DCs. TLR3 might be largely engaged in this process, which can recognize viral double-stranded RNA in birds (Chen et al., 2013). However, we also observed that chBM-DCs expressed low levels of the maturation markers CD86 and CD40 in response to IBDV. We inferred that the direct infection of DCs by IBDV may trigger a viral scape mechanism to
J. Liang et al. / Veterinary Immunology and Immunopathology 164 (2015) 51–55
impair DC function and impede the initiation of an effective immune response. Previous researchers reported that IAV-infected human myeloid DCs were impaired in their ability to cross-present exogenous antigen via MHCI and further inhibit DCs maturation (Waithman and Mintern, 2012). Ruth Chin et al. also have previously shown that the different viral strains infection could produce different proteins and RNA within human DCs. Such virus strain differenced in infection of human DCs could account for the different capacities to stimulate DCs maturation (Chin et al., 2013). In this regard, we deduce that the IBDV strain infection specifically inhibited chBM-DCs maturation. It is well known that only DCs are capable of activating naïve T cells and mature DCs have a strong capacity to induce proliferation of allogeneic T cell (Harris and Fabry, 2012). We therefore characterized their ability to drive strong allogeneic MLR when mature. In our studies, inactivated-IBDV or IBDV stimulated-DCs could dramatically evoked the ability to stimulate proliferation of naive T cells in the allogeneic MLR, suggesting that the chBMDCs took up and processed these viruses for presentation to T cells in vitro (Nierkens et al., 2013). Compared to IBDV, inactivated-IBDV had better effect on stimulating primary T cell responses and this might be correlated well with the higher expression of the co-stimulatory molecules CD40 and CD86 on inactivated-IBDV stimulated-DCs. Another reason for the lower T-cell proliferation of IBDV-stimulated DC is that the altered response was dependent on the viral protein 2 and did not require infection of DC with IBDV (Azizi et al., 2013). Other reasons for the dose-dependent proliferative response may be that properties of DC that contribute to successful T-cell activation (Chen et al., 2013). In conclusion, we successfully cultured chBM-DCs which had been characterized in terms of their morphology and high purity in vitro. We demonstrated that inactivated-IBDV in contrast to live IBDV was better to induce phenotypical and functional maturation of chBMDCs. This is the first report that inactivated IBDV is strong inducers of chBM-DCs maturation. These results suggest a novel mechanism to explain why infection of live IBDV fails to trigger an effective specific immune response or develop immune memory. Exploring the mechanisms involved in enhancement of the immune response by IBDV may greatly accelerate vaccine development. Conflict of interest statement There were no potential conflicts (financial, professional or personal) that are relevant to the manuscript. Acknowledgments This work was supported by the National Science Grant of P. R. China (No. 31172302) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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