The thymus is the specific organ in which the maturation and differentiation of avian T-lymphocytes take place. T lymphocytes, or T cells, are of a key importance to the immune system and are at the core of adaptive immunity. Chickens infected with MDV display thymic atrophy (Figure
1A) and eventually the formation of T-cell lymphoma. However, little is known about the underlying mechanisms of this phenomenon. In the present study, 119 proteins differentially expressed in thymus specimens from chickens infected with MDV were identified by two-dimensional electrophoresis and mass spectrometry at seven time points. The chicken thymuses displayed severe atrophy at 21, 28 and 35 days after infection with RB1B, and their size gradually returned to normal at 42 dpi. The change in thymus size during the course of the MDV infection may have influenced the expression of cellular structural proteins, enzymes and cytoskeleton proteins. However, we should also note that the 2-DE and real-time PCR analyses were conducted using the same amounts of protein and mRNA, which might exclude the effect of thymus size to some extent. In addition, the change in the cellular composition of the thymus could be affected the total proteome. The thymic atrophy in the chickens infected with MDV has a significant influence on host immune suppression. Permanent immunosuppression tends to correlate with the eventual development of tumours, which enhances our understanding of the mechanisms of T-lymphoma formation.
To better understand host responses to MDV infection, the expression of viral genes (gB and Meq) was detected using real-time PCR (Figure
6). We found a good correlation between viral gene product and host differentially expressed proteins. The expression levels of gB and Meq were transiently increased at 4 and 7 dpi and decreased at 14 dpi during the latent infection period. The gB and Meq expression levels were again increased at 21 dpi, suggesting the occurrence of MDV reactivation and transformation (Figure
6). At 21 dpi, the over-expressed viral gene products led to serious changes in protein expression levels and thymic atrophy (Figures
6). These changes could be due to the disease pathology induced by MDV infection. Consistent with the MDV pathogenesis, early cytolytic infection occurred at 3 to 7 dpi and then entered latency, followed by the proliferative/transformation phase, leading to lymphoma formation at approximately 21 to 28 dpi. After 28 dpi, the mRNA levels of gB and Meq in the MDV-infected chickens displayed a rapid decrease, and little mRNA was detected at 42 dpi. This change may be due to the cellular response of lesion regression. It has been reported that lymphomas can occur at any time from approximately 3~4 weeks, and lesion regression can occur after lymphomas
More interestingly, more than 20 differentially expressed proteins were directly associated with immunity, apoptosis, tumour development and viral infection and replication (Table
1). Notably, at least nine proteins were identified for the first time in this study: macrophage migration inhibitory factor, heat shock protein 90-alpha, annexin A1, far upstream element-binding protein 1, septin-6, septin-9, beta-galactoside-binding lectin, mCG49244 and an unnamed protein product. Among these proteins, some formed a network map encompassing TP53, MYC and HIF1A at its core and are directly associated with immunity, apoptosis, tumour development and viral infection and replication (Figure
4). Within this map, we found that MIF, HSP90AA1, NPM1, STMN1(OP18) and VIM can interact with the tumour suppressor protein TP53. It has been reported that the Meq oncoprotein directly interacts with p53 and inhibits p53-mediated transcriptional activity and apoptosis
. This scenario could provide an interesting link between MEQ and these proteins and enhance our understanding of MDV pathogenesis. The mRNA levels of these proteins were confirmed by real-time PCR (Figure
5), and some of these proteins are further discussed in this study.
Roles of the differentially expressed proteins in MDV infection
After MDV infection, the expression of the MIF protein displayed a slight increase at 4 dpi, followed by a down-regulation and subsequent increase at 42 dpi. The mRNA expression level of MIF was down-regulated at all stages, which may have resulted for two reasons. First, different mechanisms control the transcription and translation of the MIF gene in chicken thymus, and the mRNA abundance is not always consistent with the protein level. Second, it is possible that the transcription level of the MIF gene is easily influenced by MDV during the early stages of the infection. However, MIF is a pro-inflammatory factor, and its translational level is enhanced by the host’s regulatory mechanisms. Notably, the differences in the expression levels of the MIF mRNA and protein could be involved in the MDV infection. It is believed that macrophages transfer the virus to B cells, which are the primary target cells that are infected between 3 and 6 days post-infection
. A heavy infiltration of lymphocytes and macrophages occurs around blood vessels at 8–10 days post-MDV infection
, and a study has demonstrated that some new MDV strains can replicate in macrophages, which leads to increased macrophage death
. MIF is involved in virus infection, and its target cells are mainly macrophages. It has been reported that human cytomegalovirus (HCMV), a herpesvirus, paralyses macrophage motility through the down-regulation of chemokine receptors, reorganisation of the cytoskeleton, and release of MIF
. MIF also promotes HIV-1 replication through the activation of HIV-1 long terminal repeats (LTR)
. Increased MIF at 4 dpi may promote MDV replication. The down-regulation of MIF indicates a strong macrophage migration activity, and macrophages that carry MDV could spread the MDV infection to other cells. Another protein, Anx-A1, was induced at 7, 21, 28 and 35 dpi and may be associated with MDV infection. Research studies suggest that the annexin family members are involved in the viral replication cycle, which may integrate the regulation of virus infection by forming networks
. Nucleolar protein B23 (NPM1), an acidic nucleolar protein, was up-regulated in the thymus of chickens infected with MDV and may indicate the nuclear importation of MDV. It has been demonstrated that NPM1 stimulates the nuclear importation of the HIV-1 Rev protein
, and NPM1 is also an important factor for the nucleolar localisation of the HIV protein Tat
. The reduction in the Hsp90alpha level during early MDV infection might be related to a latent infection. Hsp90 plays an important role in the replication and infectivity of some herpesviruses, such as herpes simplex virus type 1
, Epstein–Barr virus (EBV)
 and Kaposi’s sarcoma-associated herpesvirus (KSHV)
, and is required for the folding, stability and intracellular transport of multiple viral proteins and for the activity of viral polymerases. Taken together, our observations suggest that changes in the levels of these proteins influence MDV replication and infection. However, the specific mechanisms are unknown and will require further study.
The chicken immune response to MDV infection
Suppression of the immune response by MDV infection is a critical feature of the disease. MIF down-regulation may contribute to the inhibition of the immune response. MIF, a classic pro-inflammatory cytokine and a pivotal regulator of innate immunity, promotes innate and adaptive immune responses through the activation of macrophages and T cells
[25, 26]. Moreover, it directly inhibits the immunosuppressive actions of glucocorticoids
 through the suppression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1)
[28–30]. MKP-1, which is induced by glucocorticoids, inactivates the proinflammatory ERK1/2, JNK, and p38 pathways. Anx-A1, a pivotal regulator of the innate and adaptive immune systems, also promotes immunosuppression
. This protein was induced at 7, 21, 28 and 35 dpi and has a powerful suppressive effect on the innate immune system. Recent investigations on the role of this protein in the adaptive immune response have revealed a previously unknown ‘dark side’ to this protein, that is, it is a positive modulator of T cell activation
. High levels of Anx-A1 influence the differentiation of T cells in vivo, and hence, may contribute to the development of T-cell-driven autoimmune diseases.
While heat shock proteins are molecular chaperones, they have also been implicated in the stimulation of the innate and adaptive immune systems
[33, 34]. Recent biochemical evidence highlights the role of Hsp90alpha in the endogenous processing of MHC class I antigens, and the absence of Hsp90alpha results in the decreased surface expression of MHC I
. Levy et al.
 reported that RB1B was able to markedly decrease MHC class I expression. In addition, those authors found that MDV pUL49.5 directly down-regulates MHC class I expression
, and beta2 microglobulin was also decreased in the bursa of Fabricius of chickens infected with RB1B
. A subsequent gene expression study indicated a down-regulation of MHC class II expression in the spleens of MDV-infected chickens
. The specific relationship between the down-regulation of HSP90A and MHC class I expression during the course of MDV infection is not yet clear. However, these findings indicate that a potential mechanism of immune evasion mediated by MHC expression on cell surfaces might be employed by MDV. In addition, the immune suppression that occurs after MDV infection is not only related to changes in immune-related proteins but may also be associated with immune evasion.
Apoptosis and thymic atrophy after MDV infection
The thymic atrophy of chickens infected with MDV is the most significant manifestation of immune suppression and might be related to apoptosis. Morimura et al. reported that MDV can induce apoptosis and the down-regulation of CD8 molecules on peripheral CD4+ T cells and in the thymus, which could contribute to immune suppression
[10–12]. In this study, although we did not detect cell apoptosis by flow cytometry, changes in some proteins indicated apoptosis in the thymuses of the chickens. According to the network of protein–protein interactions in the thymuses infected with MDV, five differentially expressed proteins can interact with P53 (Figure
6). The decreased levels of stathmin/oncoprotein18 (Op18) and MIF may increase the level of p53 and promote p53-mediated apoptosis before T-cell lymphoma formation. It has been demonstrated that MIF suppresses the expression of p53 and its activity
[26, 38]. MIF increases resistance to apoptosis by activating the nuclear factor-kappa B (NF-κB) system and repressing the function of p53
. Changes in Op18 may be related to the increase in P53 because p53 is associated with the negative regulation of stathmin expression
[40–42]. In the thymus, this protein was mainly down-regulated and was significantly differentially expressed at 21, 28 and 35 dpi. Lu et al.
 also reported that Op18 was significantly reduced at 4, 7 and 21 dpi in the bursa of Fabricius of chickens infected with RB1B. In addition, ANX-A1 is relevant to the regulation of cell growth and apoptosis
[43, 44], and ANXA1 overexpression has been shown to promote apoptosis
[45, 46]. It has also been reported that ANXA1 expression in leukaemic cells mediates the engulfment of apoptotic cells by macrophages
. In short, MDV infection results in immune suppression and the induction of apoptosis, which eventually leads to thymic atrophy.
T-cell lymphoma formation during MDV infection
The Meq oncoprotein of the Marek’s disease virus is the major oncogene involved in the induction of tumours and inhibits p53 transcriptional and apoptotic activities by interacting with p53
. Permanent immunosuppression tends to correlate with the eventual development of tumours. The role of host proteins associated with tumour growth and metastasis in the formation of T-cell lymphoma cannot be ignored. MIF, a negative regulator of the important tumour suppressor p53, is involved in tumour occurrence and evolution
[48–50]. A significant amount of evidence indicates that MIF influences several important biological mechanisms and processes by which tumours thrive and spread. One of the most important of these mechanisms is the modulation of hypoxic adaptation within the tumour microenvironment through the direct promotion of the hypoxia-induced stabilisation of HIF-1α
. We speculate that the increased level of MIF protein observed at 42 dpi may indicate hypoxic adaptation within the tumour microenvironment. Anx-A1 is directly related to tumour development
. This protein was induced at 7, 21, 28 and 35 dpi and could be a key host factor that enhances the formation of T-cell lymphomas. Strong evidence for this process is provided by the finding that the increased expression of Anx-A1 promotes tumour growth, invasion and metastasis in gastric carcinoma
, breast cancer
 and colorectal cancer
. Using AnxA1- knock out (KO) mice, it has been determined that tumour growth and metastasis are significantly decreased, whereas rodent survival and tumour necrosis are significantly increased when tumours grow in AnxA1-KO mice
. In addition, the up-regulation of several cytoskeletal network proteins, e.g., vimentin, beta-actin and keratin (type I cytoskeletal 15), also promotes tumour growth and metastasis. Research suggests that vimentin, a major intermediate filament (IF) protein of mesenchymal cells, is very important for tumour growth and metastasis
[58, 59]. Beta-actin specifically controls cell growth and migration
, and an increase in beta-actin levels correlates with a higher level of invasiveness for a select hepatoma in Morris 5123 cells
. Changes in these proteins indicate that MDV infection and T-cell lymphoma formation involve the host cytoskeleton. Nucleolar protein B23 (NPM1) plays multiple roles in cell growth and proliferation
. Thanthrige-Don et al. also reported an up-regulation of beta-actin and NPM1 in the spleens of MDV-infected chickens
. Interestingly, a report has shown that the interaction between Meq and Hsp70 is significant during MDV oncogenesis
. However, the biological consequences of the Meq–Hsp70 interaction are not clear. Finally, HSP90A was reduced at 21 dpi; however, whether HSP90 can interact with Meq and affect Meq carcinogenicity remains to be studied.
In addition, septin 9 and septin 6 levels were significantly decreased in the thymuses of chickens infected with MDV. Septins are a highly conserved family of GTP-binding cytoskeletal proteins implicated in oncogenesis
[64, 65]. Septin 9 (SEPT9), a DNA methylation-based biomarker, has been functionally linked with oncogenesis through its activation of the hypoxia-inducible factor-1 (HIF-1) pathway, which promotes tumour progression, and the c-Jun-N-terminal kinase (JNK) pathway, which plays an important role in cell proliferation, cell transformation, and tumour progression
[66, 67]. The differential expression of heterogeneous nuclear ribonucleoproteins (hnRNPs), including HnRNP A / B, HnRNP A2/B1, HnRNP H, and HnRNP C1/C2, was also detected in this study. These proteins play key roles in tumour development and progression
. Studies have shown that hnRNP A2 / B1 and hnRNP A1 can combine their telomere DNA sequences and that their encoded protein isoforms can interact with the telomerase. Tumour cells fail to enter senescence due to their telomere lengths, which are maintained by telomere-bound proteins that recruit the enzyme telomerase
[68, 69]. Currently, the roles of septins and hnRNPs in MD tumour progression are not clear and require further study.