Differential expression of pro-inflammatory and anti-inflammatory genes of layer chicken bursa after experimental infection with infectious bursal disease virus

Zhi-Yong Xu,∗,1 Yan Yu,∗,1 Yan Liu,† Chang-Bo Ou,∗ Yan-Hong Zhang,∗ Ting-Yu Liu,∗ Qiu-Xia Wang,∗ and Jin-You Ma∗,2 ∗

College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang 453003, China; and † Army Eighty-three Group Army Hospital, Xinxiang 453000, China cytokine IL-10 in the infected group increased to varying degrees at 12 to 192 h, respectively. Furthermore, the IL-1β mRNA expression peaked at 48 h; the mRNA transcript levels of IL-6, IL-8, and IL-10 were the highest at 96 h; TNF-α mRNA expression peaked at 120 h; the IL-7 mRNA expression peaked at 144 h; and the TGF-β mRNA transcript level was the highest at 192 h. Taken together, these observations indicated that along with the change pattern of IBDV proliferation in BF, the mRNA expression of cytokines (IL-1β , IL-6, IL-7, IL-8, IL-10, TNF-α, TGF-β ) obviously increased, and the kinetics of each of these cytokines was different. The kinetics of IL-6/IL-10 mRNA expression ratio was significantly positively correlated with that of the virus load. These results suggest that IBDV infection seriously interferes with the natural immune response mediated by inflammatory cytokines in chickens.

ABSTRACT Infectious bursal disease (IBD) is one of the most prevalent infectious diseases caused by IBD virus (IBDV), which results in bursal necrosis and immunosuppression that cause severe damage to the immune system in chickens. Cytokines are important mediators and regulators of both types of host responses. In the present study, layer chickens were artificially challenged with IBDV, and the differential expression of inflammatory genes was explored by using quantitative real-time PCR, which offered basic data for further study of IBDV pathogenesis. Data showed that after IBDV infection, the virus load in the bursa of Fabricius (BF) peaked at 96 h and then gradually decreased. Compared with those of the negative-infected group, the mRNA expression levels of pro-inflammatory cytokines (interleukin [IL]-1β , IL-6, IL-7, IL-8, tumor necrosis factor [TNF]-α, transforming growth factor [TGF]-β ) and anti-inflammatory

Key words: anti-inflammatory gene, gene expression, infectious bursal disease virus, pro-inflammatory gene, VP3 2019 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pez312

INTRODUCTION

amassed on the antigenic and immunological characteristics of the virus, the disease has not yet been brought fully under control. Avian cytokines, similar to their mammalian counterparts, are influential in host immune response to pathogenic infection (Kaiser and Stabeli, 2008). During viral infection, transforming growth factor beta (TGF-β ); tumor necrosis factor-α (TNF-α); and interleukin 6, 7, 8, 10, and 1 beta (IL-6, IL-7, IL-8, IL-10, IL-1β ) are common inflammatory factors (Eldaghayes et al., 2006; Klemens et al., 2007; Raj et al., 2011). IL-10, an anti-inflammatory cytokine, can contribute to the clearance of the pathogen and facilitate antigen acquisition from dead cells for cross-priming activated antigen-presenting cells (Kelly et al., 2001; Ohsuzu, 2004). TGF-β , IL-8, TNF-α, IL-6, and IL-1β , which are pro-inflammatory cytokines, can induce the transcription of different target genes that function in differentiation, proliferation, and activation of many immune cells and are involved in the innate

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a bi-segmented and doublestranded RNA virus and is a major avian pathogen responsible for the immunosuppressive disease affecting young chickens from 3 to 6 wk of age. Infectious bursal disease virus targets the chicken’s immune system in a remarkably comprehensive and complex manner by destroying B lymphocytes, attracting T cells, and activating macrophages. Infectious bursal disease results in economic loss due to mortality, increased usage of antibiotics, and reduction in production efficiency (Yilmaz et al., 2019). In spite of all the information  C 2019 Poultry Science Association Inc. Received September 24, 2018. Accepted May 19, 2019. 1 Zhi-yong Xu and Yan Yu are joint first authors 2 Corresponding author: [email protected]

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Differential expression of pro-inflammatory and anti-inflammatory genes of layer chicken bursa after experimental infection with infectious bursal disease virus

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MATERIAL AND METHODS Experimental Animals The principles of laboratory animal care were followed, and all procedures were conducted following the guidelines established by the Animal Care and Use Committee of China. Every effort was made to minimize suffering. This study was approved by the Animal Experiment Committee of Henan Institute of Science and Technology. A total of 140 embryos of specific pathogen-free White Leghorn chickens were purchased from Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China). After hatching, the chickens were maintained in negative-pressure isolators (GJ-1, Suzhou Fengshi Laboratory Animal Equipment Co., Ltd.) for 19 D at a specific pathogen-free animal laboratory. Specific pathogen-free chickens (purchased from Merial Vital Laboratory Animal Technology Co., Ltd., Beijing, China) were given feeds and boiled water ad lib.

Virus The classic virulent IBDV strain BC6/85 that was used in the current study was purchased from China Institute of Veterinary Drug Control (Beijing, China).

Experimental Protocol The chicks were bred for 19 D and randomly divided into 2 groups reared in negative-pressure isolators (each isolator can hold up to 30 chickens). One group (n = 30+30) of chicks fed in 2 negative-pressure isolators in 1 room was inoculated with the virulent IBDV strain BC6/85 at a dose containing 106.23 EID50/0.1 mL each through the ocular–nasal route. The other group (n = 30+30) of chicks in 2 negative-pressure isolators in another room were treated with 0.1 mL of sterile phosphate-buffered saline (PBS, pH = 7.38) and kept as negative-infected controls. At 0, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h post-infection, 6 chicks (3+3; 3 chickens were taken from each isolator) were randomly selected in each group and euthanized via cervical dislocation under deep Nembutal anesthesia (45 μg/g of BW, intraperitoneal injection; Shanghai Chemical Factory, Shanghai, China) for sample preparation. During necropsy, the bursa of Fabricius (BF) was quickly collected from each chick, then used for bursal index [bursal index = weight of bursa (mg) × 1000/body weight (g)], and finally snap-frozen in liquid nitrogen. One part of each BF was used for viral load and fixed in 4% paraformaldehyde (Solarbio, P1110, Beijing, China) for immunohistochemical staining. In every immunohistochemistry slide, the follicular areas were observed. Approximately 78 to 92 follicles per sample were observed under the quadruple lens, and the similarly sized follicles of the same number from 2 groups were selected and calculated. The other samples were used for gene expression analysis of inflammatory cytokines. Gray value is a measurement method that reflects the viral protein expression content. The mean gray value of the viral load was analyzed using the Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), in which a high gray value indicated low expression levels (Ma et al., 2016). The negative-infected BF tissue was used as parallel control. The experiment was repeated 3 times.

Immunohistochemistry Staining For immunohistochemistry staining, the sections were incubated overnight at 4◦ C with mouse antiIBDV VP3 antibody (1:200, Biorbyt, orb23535, SF, CA, USA). Next, the sections were rinsed in 0.01 M PBS (pH 7.4) and incubated with biotinylated goat anti-mouse secondary antibody (1:200, ZSGB-Bio, ZB-2305, Beijing, China) for 2 h at room temperature. After washing, the immunoreactivity was visualized via incubation in 0.01 M PBS containing 0.05%

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immune response (Hedges et al., 2000; Massagu´e, 2012; Shinji et al., 2010). IL-7, an essential survival factor, plays a critical role in the homeostasis of the immune system (Huo et al., 2016; Yeon et al., 2017). IL-1β is a pro-inflammatory cytokine that exerts multiple roles in both physiological and pathological conditions (Arranz et al., 2017). A previous study has shown that IBDV activates the T cells in the bursa of chickens and upregulates the expression levels of IL-1β and IL-6 (Liu et al., 2010). However, another study found that IL-1β may be downregulated temporarily after chickens infected with IBDV (Jones et al., 2005). A high viral load in the bursa is associated with significantly high expression levels of cytokine genes (Abel et al., 2005; Abdul-Careem et al., 2008). Furthermore, the change in cytokine expression level is closely associated with organizational destruction, inflammation, and apoptosis (Rautenschlein et al., 2002). The study found that the bursa is the target organ of viral replication, and peak virus titers in the bursa can be detected between 3 and 5 D after IBDV infection (Wang et al., 2011). Cytokines are important mediators and regulators of host responses against foreign antigens, and their main function is to orchestrate the functional activities of the cells of the immune system (Liu et al., 2010). However, cytokine responses to IBDV in the bursa of chickens are poorly understood. For example, following IBDV challenge, TNF-α, and IL-7 expression in the chicken bursa has not been studied, and IL-1β expression trends were controversial. Moreover, the relationship between inflammation factors and IBDV load has not been reported until now. Thus, the present study aimed to systematically investigate the dynamic expression of inflammatory cytokines during IBDV infection and offer basic data for studying the balance of inflammatory cytokines after viral infection.

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Table 1. Sequence of the primers used in real-time PCR. Gene

Reverse primer 

5 -GGACTATTTTCAATCCAGGGACG-3 5 -TGCCACTTCTCCTTGTTCTGTCG-3 5 -TGCGTCCTCTTTACATTGACTTCC-3 5 -ATGTCGTGTGTGATGAGCGGC-3 5 -AAATCCCTCCTCGCCAATCTG-3 5 -GCTGCTCTGTCGCAAGGTAGG-3 5 -ACTATCCTCACCCCTACCCTGTC-3 5 -TATTGCTGCGCTCGTTGTTGAC-3



5 -GGGCAGGACCTCATCTGTGTAG-3 5 -TCTTGCAGCATCTGTCACGATATTC-3 5 -CGTGCTGTGAGTGGTGTCTGC-3 5 -AGGCGGTAGAAGATGAAGCGG-3 5 -CCTCACGGTCTTCTCCATAAACG-3 5 -ACATCTTGAATGGATTTAGGGTGG-3 5 -GGTCATAGAACAGCACTACGGG-3 5 -GATACCTCTTTTGCTCTGGGCTTC-3

3 ,3-diaminobenzidine tetrahydrochloride (D5637, Sigma-Aldrich, St. Louis, USA) and 0.003% hydrogen peroxide for 3 min in the dark. After the final rinse, the sections were mounted. The specificities of immunostainings were verified by omitting the primary antibodies from the first incubation. The viral loads were calculated by relative quantification to negative-infected controls.

Quantitative Real-Time PCR Total RNA was isolated from BF with TRIzol reagent (Life Technologies, 15596018, Carlsbad, USA) following the manufacturer’s protocol. DNA degradation of the total RNAs and first-strand cDNA synthesis were performed using PrimeScriptTM RT reagent kits with gDNA eraser (TaKaRa, RR047A, Dalian, China) according to the manufacturer’s instructions. Several pairs of primers were designed for the detection of proinflammatory and anti-inflammatory genes. β -actin was applied as internal standard. The primer pairs for inflammatory genes and β -actin are shown in Table 1. Experiments on each sample were performed in triplicate with the above primers. Real-time PCR was performed briefly as follows: an initial incubation for 30 s at 95◦ C, 40 cycles of 5 s at 95◦ C, 20 s at 58◦ C, and 20 s at 72◦ C. Quantification was based on the fluorescence detection R by using real-time PCR with SYBR Premix Ex TaqTM II (TakaRa, RR820A, Dalian, China). The specificities of the PCR products were assessed using dissociation curve analyses. After PCR, data were analyzed with an ABI 7300 instrument (Applied Biosystems, Marsiling, Singapore) by using the 2−ΔΔCT method.

Statistics Data were analyzed using the software package SPSS 13.0 for Windows. Results were expressed as mean ± standard error. Two-tailed 1-way ANOVA was employed for all statistical analyses. SPSS Pearson correlation analysis was used to analyze the correlation of dynamic changes of pro-/anti-inflammatory gene expression ratio and the variation trend of VP3 expression. Analyses of the correlation between the dynamic changes of pro-/anti-inflammatory gene expression ratio and the variation trend of VP3 expression were performed using Pearson’s correlation coefficient; r > 0

Figure 1. Dynamic changes of the chickens’ bursa index after infection with infectious bursal disease virus on 0 to 192 h. Comparing with Mock-infected group. ∗ P < 0.05 indicate that the difference is significant, ∗∗ P < 0.01 indicate that the difference is highly significant, and the following figures are the same.

and r < 0 indicate positive and negative correlations, respectively; P < 0.05, P < 0.01, and P > 0.05 indicate that the difference is significant, highly significant, and not significant, respectively (Ma et al., 2016).

RESULTS AND DISCUSSION Infectious bursal disease virus is a considerably severe viral disease that causes immunosuppression in chickens accompanied with other pathogen infections. Infectious bursal disease virus BC6/85 is a classical Chinese virulent strain and can cause immunosuppression without or with low mortality (Wang et al., 2011). In the present study, the bursal growth index was calculated at 0 to 192 h between the negative-infected group and infected group (Figure 1). The results showed that the bursal index of chickens in the infected group remarkably decreased at 24 h (4.23), and the trend remained the same until the end of the experiment at 192 h (1.21). However, the bursal index of chickens in the negativeinfected group did not show significant changes and almost remained the same (i.e., from 4.09 to 4.86). The bursal index in this study was consistent with that of previous studies (Ou et al., 2017a,b), and these data showed that the model was successful. The IBDV genome encodes an RNA-dependent RNA polymerase (VP1), 2 major structural proteins (VP2 and VP3), a viral protease (VP4), and a nonstructural protein (VP5). On the basis of VP3 analysis, IBDV replication complexes were localized to vesicular

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IL-10 IL-7 TGF-β IL-1β IL-6 IL-8 TNF-α β -actin

Forward primer 

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(a)

72h

96h

120h

Figure 2. Dynamic Expression of IBDV VP3. (a) Dynamic changes of the infectious bursal disease virus (IBDV) VP3 gray value at 0 to 192 h. (b) Immunohistochemistry staining to detect the IBDV VP3 expression levels at 72, 96, and 120 h, respectively. Magnification 400×.

structures bearing features of early and late endocytic compartments, interfering with the endocytic pathway. VP3 plays a central role in ensuring the viability of the IBDV replication cycle by preventing programmed cell death (Busnadiego et al., 2012). In the current study, compared with that of the negative-infected group, the VP3 gray value of the infected group did not significantly differ (P > 0.05) at 0 h and dramatically (P < 0.01 or P < 0.05) decreased at 12 to 192 h, respectively (Figures 2a and b), which indicated that the VP3 expression of the infected group obviously increased. Furthermore, the VP3 expression of the infected group BF peaked at 96 h and then slowly dropped until the end of the experiment (Figure 2), which indicated that the viral load of BF peaked at 96 h and then gradually decreased. The mRNA transcript levels of pro-inflammatory cytokines (IL-6, IL-7, IL-8, IL-1β , TGF-β , and TNF-α) in chicken bursa were quantified by using real-time PCR. The results showed that compared with that of

the negative-infected group, the IL-8 mRNA expression level in the infected group dramatically increased (P < 0.01 or P < 0.05) at 12 to 192 h. Moreover, the relative mRNA expression of IL-1β , IL-6, and TNF-α increased remarkably (P < 0.01 or P < 0.05) at 12 to 144 h, and TGF-β mRNA expression levels increased dramatically (P < 0.01 or P < 0.05) at 24 to 192 h (Figure 3). The mRNA expression level of IL-7 was rapidly reduced from 12 to 24 h and then obviously increased (P < 0.01 or P < 0.05) at 48 to 192 h. Furthermore, the IL-1β mRNA expression peaked at 48 h, which was 8.76-fold higher than that of the negativeinfected group; the mRNA expression levels of IL-6 and IL-8 were the highest at 96 h, which were 62.36- and 133.54-fold higher than those of the negative-infected group, respectively. The TNF-α mRNA expression peaked at 120 h, which was 2.58-fold higher than that of the negative-infected group. The IL-7 mRNA expression level peaked at 144 h, which was 6.90-fold higher than that of the negative-infected group. The TGF-β

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(b)

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mRNA expression level was the highest at 192 h, which was 9.09-fold higher than that of the negativeinfected group. According to a previous report, a strong overexpression of IL-6 and IL-8 was observed

in IBDV-infected chicken bursa at 3 D (Carballeda et al., 2015). The IBDV infection depleted lymphocytes in the medullary area and significantly stimulated the relative expression of IL-6 mRNA of the bursa

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Figure 3. Dynamic change of inflammatory factors IL-10, TGF-β , IL-7, IL-1β , IL-6, IL-8, and TNF-α mRNA transcript levels at 0 to 192 h.

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(P < 0.05) compared with that of the uninfected bursa (Long et al., 2011). The expression of chemokine gene IL-8 was also higher in cIBDV-infected chickens during the early phase of infection (Rauf et al., 2011). In the IBDV-infected commercial chickens, IL-8 was significantly increased compared with that of the uninfected commercial chickens (Raj et al., 2011). In addition, chickens infected with virulent IBDV (IM strain) exhibited a strong upregulation of IL-1β (65-fold) at 3 D post-infection (dpi) in bursal macrophages (Khatri et al., 2005). TGF-β is a potent immunosuppressive cytokine regulating immune homeostasis (Akhurst and Hata, 2012). A previous study showed that IBDV could stimulate TGF-β expression in chicken lymphoid tissues, which likely promotes IBDV-induced immunosuppression in chickens (Eldaghayes et al., 2006). The TGF-β mRNA level in the bursa following IBDV infection was markedly upregulated (Jain et al., 2013). In the present study, IBDV considerably improved the IL-6, IL-8, and TGF-β gene transcription levels, which were consistent with those in previous reports (Long et al., 2011; Raj et al., 2011; Akhurst and Hata, 2012). Furthermore, compared with those of uninfected commercial chickens, the IL-1β expression levels in the IBDV-infected commercial chickens were significantly increased (Raj et al., 2011). Unexpectedly, IL-1β was not strongly upregulated (i.e., only a negligible upregulation was observed in the spleen of IBDV-inoculated chickens at 1 and 3 dpi) (Jain et al., 2013). The present study showed that IBDV obviously improved the IL-1β gene transcription level, which was in agreement with the previous reports (Khatri et al., 2005; Liu et al., 2010; Raj et al., 2011). IL-7, an important cytokine, can facilitate the recovery of B cell number after immunosuppression (Milne and Paige, 2006). Human Pegivirus significantly promoted the composition of IL-7 (Blackard et al., 2017). After infection with hepatitis B virus, the IL-7 level was obviously increased (Zhong et al., 2016). Infectious bursal disease virus inhibited the mRNA expression of IL-7 from 12 to 24 h. However, it obviously promoted the IL-7 mRNA expression at 48 to 192 h in the

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Figure 4. Dynamic changes of IL-6/IL-10 relative expression ratio at 0 to 192 h.

chicken bursa, which was similar to previous reports on other pathogens (Zhong et al., 2016; Blackard et al., 2017). Another study found that the cells infected with human immunodeficiency virus type 1 express the TNF-α (Moyo et al., 2017). In the current study, IBDV obviously improved the TNF-α gene transcription level in the chicken bursa, which was similar to a previous report concerning other pathogens (Moyo et al., 2017). During the early and middle phases of IBDV infection, IL-6, IL-8, IL-1β , and TNF-α relative mRNA expression levels were the most prominent, which indicated that these cytokines were the main proinflammatory factors causing bursal inflammation in the early and middle infection phase. The IL-7 relative mRNA expression was the most significant in the late infection phase of IBDV, indicating that this cytokine had a significant effect on bursal inflammation in the late infection stage. In addition, the TGF-β family is the main stimulator leading to conjunctival scarring post trabeculectomy, and various cells, such as fibroblasts, and macrophages, can secrete TGF-β (Razzaque et al., 2003). Infectious bursal disease virus induces bursal tissue fibrosis in the late stage of infection (Zhao et al., 2016). In the present study, the relative mRNA expression of TGF-β was the most significant in the late phase of IBDV infection. This finding indicated that TGF-β might play a complicated and important role in bursal tissue fibrosis in the late stage of infection, which is associated with disease progression in IBDV-infected chickens (Zhao et al., 2016). Real-time quantitative PCR analysis also revealed that the expression level of anti-inflammatory cytokine IL-10 was significantly upregulated following IBDV challenge. A previous study reported that the IL-10 mRNA level in the bursa following IBDV infection was markedly upregulated (Raj et al., 2011). The IL-10 transcript was upregulated at 5 dpi with IBDV (Liu et al., 2010). Furthermore, IBDV caused a significant increase in IL-10 mRNA level (Yu et al., 2015). In the present study, IBDV obviously increased IL-10 expression during the infection phase. This finding is consistent with the fact that IL-10 plays a dual role in infectious diseases (Mocellin et al., 2003) and is in agreement with previous reports (Raj et al., 2011; Yu et al., 2015). A previous study revealed that higher than normal ratio of pro-inflammatory and anti-inflammatory factors promotes pathological changes in tissues (Li et al., 2011), and a higher viral load in the bursa is associated with significantly high lesion scores of bursa (Zhao et al., 2016). Our observations suggest that the ratios of pro-inflammatory and anti-inflammatory factors were higher than the normal values throughout the infection. Furthermore, the IL-6/IL-10 ratio of infected group was obviously higher than that of negative-infected group at 12 to 120 h, and dynamic change of IL-6/IL-10 relative expression ratio was significantly negatively correlated with the variation trend of VP3 gray value

INFECTIOUS BURSA DISEASE AND INFLAMMATION

ACKNOWLEDGMENTS We are grateful to Ying Zhao, Xiaoxiao Liu, and Shanming Hu, undergraduates of College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, for their assistance in the process of experiment. The research was supported by Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No. 18IRTSTHN019), National Natural Science Foundation of China (No. 31602024), Key Science and Technology Program of Henan Province (No. 162102110038) and Science and Technology Projects of Xinxiang city (No. CXGG16026).

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(r = −0.642, P = 0.045). These results indicated that IBDV load could be estimated via detecting the change of IL-6/IL-10 relative expression ratio, and the inflammatory status might play a complicated and important role in tissue damage induced by IBDV. However, further study is necessary to elucidate its specific role. In conclusion, the current data showed that along with the challenge of different IBDV load, the IL-1β , IL-6, IL-7, IL-8, TNF-α, TGF-β , and IL-10 mRNA transcription levels were increased to varying degrees; the pro-inflammatory and anti-inflammatory factor ratios were higher than the normal values during the course of IBDV infection. The variation trend of IL-6/IL-10 relative expression ratio was significantly negatively correlated with the change of VP3 gray value. Taken together, IBDV infection seriously interferes with the natural immune response mediated by inflammatory cytokines in chickens.

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