Lyn inhibited BCR signal pathway mediates B-cell anergy induced by avian leukosis virus subgroup J

Background: Immune tolerance induced by retrovirus is a prerequisite for tumorigeness. We had reported that B-cell anergy was the main reason for immune tolerance induced by avian leukosis virus subgroup J (ALV-J). However, the molecular mechanism remains unclear. Results: Initially, we found that Lyn showed down-regulation in chick embryo broblasts (CEF) and up-regulation in B-cells infected by ALV-J. So, we speculated that tyrosine kinase Lyn plays a key role in B-cell anergy induced by ALV-J. Confocal laser scanning microscopy (CLSM) and co-immunoprecipitation (Co-IP) results demonstrated that ALV-J indirectly regulated the expression of Lyn. To further investigate the role and regulatory mechanism of Lyn in B-cell anergy induced by ALV-J, the expression levels of Lyn and Syk at different phosphorylation site, the Ca 2+ mobilization, and the expression levels of NF-κB p65 protein in vitro and vivo were detected in B-cells. The result showed that Ca 2+ mobilization was delayed and p65 expression level was decreased in B-cells after ALV-J infection. Consistently, the retrieve of Ca 2+ mobilization, expression levels of NF-κB p65 were found after RNA interference of Lyn. Subsequently, we demonstrated that the activation of phosphorylated Lyn protein at Tyr507 site played a critical role in B-cells anergy, which were veried by the fact of the signicantly up-regulation of the expression levels of phosphorylated Syk protein at Tyr525/526 site when RNA interference for Lyn were performed in B-cells. Furthermore, immunohistochemical (IHC) staining results conrmed that the expression levels of Lyn phosphorylated protein at Tyr507 site in bursal cells were increased, while the expression levels of Syk phosphorylated protein at Tyr525/526 sites were decreased. Conclusions: These ndings suggested that Lyn inhibited BCR signal pathway mediates B-cell anergy under ALV-J infection(cid:0)which will provide a new insight for revealing the molecular mechanism of immune tolerance induced by ALV-J. embryonic broblasts; Co-IP: co-immunoprecipitation; CLSM: confocal laser scanning microscopy; FCM: ow cytometry; FITC: uoresceinisothiocyanate; HBV: hepatitis B virus; HIV: human immunodeciency virus; iTRAQ: isobaric tags for relative and absolute quantication; IHC: immunohistochemistry; ITAM: immunoreceptor tyrosine-based activation motif; LC-MS/MS: liquid chromatography-tandem mass spectrometry; NF-κB: nuclear factor-κB; TCID 50 : 50%tissue culture infective dose; PVDF: qPCR: quantitative real-time polymoerase chain reaction; shRNA: short-hairpin RNA; SFK: Src family kinase; SPF: specic pathogen free; WB: western blot.


Background
Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus, characterized by induction of immune tolerance and neoplasia [1][2][3]. Chickens infected with ALV-J often show persistent viremia due to the lack of effective neutralizing antibody in vivo [4,5]. The adaptive immune system is tasked with producing antibodies that recognize and eliminate a wide scope of pathogens. Obviously, the acquired immunity system often fails to exert the role of neutralizing virus in the process of ALV-J infection [6]. Although the suppressed effect of ALV-J on immune system of chickens has been studied extensively [7,8], little was still known about the molecular pathogenesis of immune tolerance.
It has been proved that the process of lymphocyte anergy is an important tolerance mechanism whereby cells are functionally inactivated [9]. Experimental data have shown that the cause of immune tolerance induced by some virus such as human immunode ciency virus (HIV) could be attributed to the anergy of lymphocytes [10]. The existence and identity of anergic B-cell in animal models or human have been known for some time [11,12], but the pathogenesis of B-cell anergy induced by some viruses has not been completely addressed. However, our previous studies has showed that the main cause of immune tolerance was associated with the B-cell anergy induced by ALV-J [13].
Previous studies have shown that the anergic B-cell sending signals through the B-cell receptors (BCR) has inherent defects, and the intracellular calcium mobilization and tyrosine kinase phosphorylation levels were also abnormal [11,14]. In contrast, BCR signaling in normal B-cell is initiated by activation of Src family kinases(SFKs)after recognition of antigen, and then that leads to transduction and propagation of BCR signals that induce expression of activation markers and prepare the cell to interact productively with T-cells [15,16]. It has been shown that strong BCR signal can cause Syk to be activated through Lyn dependent pathway, but the negative regulatory function of tyrosine kinases Lyn can induce B-cell anergy [17,18].
Here, to further study the molecular pathogenesis of B-cell anergy caused by ALV-J, the differential expression proteins in host cells of ALV-J were rstly screened by proteomic analysis, and then the molecular mechanism of how key proteins regulated BCR signal transduction and thus induce B-cell anergy under ALV-J infection was further studied.

Result
Lyn was signi cantly down-regulated in CEF infected with ALV-J To screen differentially expressed proteins which plays a key role in cell signal transduction under ALV-J infection, the TMT-based proteomic analysis and hierarchical clustering method were used to visualize changes in the abundance of differentially expressed proteins in the CEF(laboratory host cells of ALV-J) between ALV-J-infected and normal groups. GO functional analysis revealed that ALV-J infection resulted in signi cant changes in protein expression associated with immune and developmental processes in CEF (Fig. 1a). We identi ed the top 19 differentially proteins associated with immune and developmental processes in CEF. Test results showed that the abundance of tyrosine protein kinase Lyn, which mediates the B-cell signal transduction, were signi cantly down-regulated in the CEF ( Fig. 1b and 1c).We further used the quantitative real-time PCR (qPCR) and the western blot (WB) to veri ed the analysis results of proteomics in CEF infected with ALV-J (Fig. 1d, 1e, and 1f).

Enhanced expression of Lyn in B-cells infected with ALV-J
A series of research data showed that Lyn plays an important role in BCR signal transduction pathway which associated with the development, differentiation, maturation, or tolerance (anergy) of B-cell [19][20][21]. Based on the proteomic results mentioned above, we investigated the Lyn expression in B-cell infected by ALV-J. Confocal laser scanning microscopy (CLSM) analysis showed that ALV-J and Lyn were both located in the cytoplasm of chicken B-cell, indicating that ALV-J directly or indirectly affects Lyn in the cytoplasm (Fig. 2a). The mRNA level and protein level of Lyn in chicken B-cells were up-regulated after ALV-J infection tested by qPCR and WB detection, different from the proteomic data of CEF (Fig. 2b, 2c, and 2d). As another molecular switch in BCR singling, tyrosine kinase Fyn also plays the active role in BCR singling regulation [22,23]. However, the expression levels of Fyn showed no signi cant difference in chicken B-cells before and after infection (Fig. 2b, 2c, and 2d). Furthermore, we also detected the expression level of Lyn in bursal B-cells in vivo. The expression levels of Lyn were increased signi cantly in B-cells of bursa of Fabricius of chicken infected with ALV-J in 14 and 28 days old tested by immunohistochemistry (IHC) and WB (Fig. 2e, 2f, 2 g, and 2 h), which was consistent with the expression level of Lyn in chicken B-cells infected with ALV-J in vitro.

Enhanced phosphorylation of Lyn but decreased phosphorylation of Syk in B-cells infected with ALV-J
Studies on the molecular structure of Lyn have shown that there are many phosphorylation sites in Lyn, two of which have the opposite effect. The phosphorylation of Tyr397 site can activates the positive regulation effect of Lyn, but the phosphorylation of Tyr507 can activates the negative regulation effect of Lyn [24]. Based on this, we further detected the expression levels of the two phosphorylation site that determined whether Lyn was activated or not. As shown the detection results of ow cytometry(FCM), although the two phosphorylated Lyn expressed signi cantly higher than the housekeeping protein after BCR signaling pathway was activated by anti IgM antibody, the Tyr525/526 phosphorylation level of Lyn downstream direct substrate Syk decreased signi cantly compared with the same enhanced phosphorylation Lyn protein( Fig. 3a and 3b). These results indicate that Lyn actually plays an inhibitory role in BCR signal transduction of chicken B-cells infected with ALV-J.
Previous studies have shown that Lyn is mainly expressed in B-cells or other some blood cells except Tcells [25]. To further verify the actual effect of ALV-J on the expression levels of Lyn in vivo, the phosphorylation level of Lyn and Syk in bursal cells of chicken were measured by IHC and WB. In accordance with the results of in vitro assay, it was found that Tyr397 and Tyr507 phosphorylation levels of Lyn increased signi cantly in bursal cells of chicken infected with ALV-J, while the phosphorylation levels of Syk decreased signi cantly (Fig. 3c, 3d, 3e, and 3f). Above results suggest that ALV-J interferes the expression and regulation of Lyn in the BCR signaling pathway.

Phosphorylation levels of Syk were retrieved after Lyn interference in B-cells infected with ALV-J
To further verify the effect of ALV-J on the expression levels of Lyn in B-cells, the shRNA interference of Lyn was performed in chicken B-cells, and the expression levels of Lyn was detected. It was found that the protein levels of Lyn were signi cantly inhibited 24 hours after Lyn interference on chicken B-cells of normal chickens, meanwhile, the protein level of Lyn in infection group chicken B-cells signi cantly decreased after Lyn interference, which was equivalent of the expression levels of Lyn in normal group chicken B-cells ( Fig. 4a and 4b). This nding indicated that the expression levels of Lyn in chicken B-cells were promoted by ALV-J. To verify whether Lyn inhibited the BCR signaling through the phosphorylation of Tyr507 under ALV-J infection, the phosphorylation levels of Syk in chicken B-cells were detected. The FCM results showed that expression levels of phosphorylation Tyr525/525 site of Syk were signi cantly retrieved (enhanced) under activated by anti-IgM antibody after 48 hours of Lyn interference ( Fig. 4c and   4d). These ndings further con rmed that Lyn performed actually the negative regulatory effect in BCR signal transduction through the phosphorylation of Tyr507 under ALV-J infection.
Ca 2+ ux and NF-κB p65 levels were retrieved after Lyn interference in B-cells infected with ALV-J Data shows that many anergy B-cells are defective in intracellular calcium mobilization and transcription of nuclear transcription factors [26]. To further analyze, the effect of Lyn on BCR signaling cascades under ALV-J infection, the Ca 2+ mobilization and protein expression of NF-κB p65 were detected in chicken B-cells under different conditions. Test results showed that the response time of intracellular Ca 2+ mobilization was delayed, the intracellular Ca 2+ ux was decreased, and the protein level of NF-κBp65 were decreased in chicken B-cells infected with ALV-J. However, the levels of the Ca 2+ ux and NF-κB p65 were retrieved in chicken B-cells infected with ALV-J after Lyn interference (Fig. 5a, 5b, 5c, and 5d). These results further indicated that Lyn played a negative regulatory role in BCR signal cascades in ALV-J infected B-cells.

Discussion
Since the phenomenon of chickens with congenital infection with ALV-J showing persistent viremia was discovered and de ned as immune tolerance, the pathogenesis of immune tolerance induced by ALV-J has been studied continuously. Because the immune tolerance caused by ALV-J, HBV, and HIV has many common features in the pathological process [27,28], it has drawn a great attraction to study the pathogenesis of immune tolerance induced by these viruses. It has been found that the damage of lymphocytes [29], the abnormal secretion of cytokines [30], the variation of viral epitopes [31], and the expression of EAV-HP gene in embryo [32] all play more or less roles in the induction of immune tolerance by ALV-J. In previous study, we demonstrated that B-cell anergy induce by ALV-J should take most of the responsibility for immune tolerance [13]. After all, the antiviral neutralizing antibodies were produced by normal B-cells. To screening the speci c protein closely related to the immune tolerance induced by ALV-J, we selected primary cells (such as CEF) rather than cell lines (such as DF-1 cell lines or chicken B-cells lines) in process of proteomic analysis for investigating the practical regulation effect of ALV-J on protein expression [33]. As expected, we screened and con rmed that Lyn, a key protein initiating BCR signal transduction in B-cells [34], was signi cantly affected by ALV-J in both CEF cells and chicken B-cells.
Tyrosine kinase Lyn is a member of SFKs expressed in various cells except for T lymphocytes, and exerts a unique dual role acting both as a positive and a negative regulatory molecule in BCR signaling [35,36]. The self-phosphorylation of Lyn protein triggers the assembly of the BCR signalosome and cascade reaction of BCR signal transduction [37]. In response B cells, BCR signaling is initiated by Lyn phosphorylating the ITAM, which further recruit and activate Syk [38]. However, anergic B-cells are characterized by reduced ability to proliferate and secrete antibody after antigen stimulation accompanied by reduced BCR-signaling responses which including delay of intracellular calcium mobilization and activation of inhibitory tyrosine kinase phosphorylation [39,40]. Antigen-induced aggregation of the BCR on normal mature B-cells results in the initiation of BCR signal cascades that culminate in proliferation, the increased of the Ca 2+ in ux and alterations in protein expression such as NF-κB p65. Our data showed that the response time of Ca 2+ mobilization in chicken B-cells was delayed, and as one of BCR signal activation events, the intracellular Ca 2+ in ux level was also signi cantly decreased. Meanwhile, the protein level of NF-κB p65 was also decreased. In this context, these results indicated that Lyn appears to function as a driver of inhibitory signaling pathways of BCR under ALV-J infection.
The unique role of Lyn in down-modulating B-cell receptor (BCR) activation mainly is mainly realized by the phosphorylation of inhibitory molecules and receptors [41]. Moreover, chronic antigen stimulation drives biased monophosphorylation of CD79 immunoreceptor tyrosine-based activation motif (ITAM) leading to recruitment of Lyn instead of Syk, which ultimately leads to B-cell anergy [42,43]. Present study results showed that ALV-J signi cantly up-regulated the protein expression level of Lyn in chicken B-cells and activated the phosphorylation of Tyr507 site in Lyn, which triggered the inhibitory effect of Lyn in BCR signal transduction, as shown by the decrease of the phosphorylation level of Syk, a downstream direct substrate of Lyn. Therefore, we suggested that the inhibitory effect of Lyn on BCR signal transduction is the crucial factor for the anergy of chicken B-cells induced by ALV-J.
In this study, the up-regulation of Lyn expression in chicken B-cells infected with ALV-J eventually led to the suppression of BCR signaling and the down-regulation of the nuclear transcription factor NF-κBp65.
We speculate that these anergic B-cells eventually undergo apoptosis and lead to the maintenance of viremia. Interestingly, previous studies have shown that up-regulated of programmed cell death protein 1 (PD-1) or down-regulated of anti apoptotic molecule Bcl-2 in immature B-cells in the peripheral blood of HIV patients nally made B-cells prone to apoptosis by endogenous apoptotic pathway [44,45], which was the important pathological process of immune tolerance induced by HIV-1.Therefore, we hypothesized that ALV-J, as a chronic stimulant antigen, activated the negative regulation role of Lyn in Bcell signal transduction.

Conclusions
These ndings suggested that ALV-J activated the negative regulation role of Lyn in B-cell signal transduction which induced B-cell anergy. Previous studies on the role of Lyn in B-cell signal transduction were mainly carried out on transgenic animal models or gene knockout cells. Here, we investigated the role of Lyn in chicken B-cell in the context of virus infection, which will provide a new insight for studying the pathogenic mechanism of immune tolerance induced by retrovirology viruses. Admittedly, further studies are necessary to investigate how ALV-J regulates the expression of Lyn.
The 6-day-old embryos of Leghorn speci c-pathogen-free (SPF) chickens (Jinan poultry technology company, China) were injected with ALV-J through the allantoic cavity to establish the infected group (n = 30).The control group (n = 30) was established by injecting DMEM instead of virus. The treatment of virus inoculation, chicken embryos incubation, and virus detection were all followed as our previous description [13].

Proteomics assay and bioinformatics analysis
In order to screen differentially expressed proteins that plays a key role in cell signal transduction under ALV-J infection,protein lysates from ALV-J-infected CEF and normal CEF were collected for proteomic analysis and bioinformatics analysis. The experimental process, iTRAQ labelled LC-MS/MS, and bioinformatics analysis were all followed as our previous description [48].

Immunohistochemistry
At the age of 14 and 28 days, the bursa of Fabricius from infected chickens and control chickens were sampled, formalin-xed, para n embedded, and sectioned (5-µm thickness) sections for IHC. All chickens were euthanized with sodium pentobarbital before the organs were removed. The perform of IHC test were followed as our previous description. [13]Negative controls were also performed with the same tissues. Primary antibodies include mouse anti-chicken Lyn monoclonal antibodies (Novus Biotech,