PD-1-Mediated PI3K/Akt/mTOR, Caspase 9/Caspase 3 and ERK Pathways Are Involved in Regulating the Apoptosis and Proliferation of CD4+ and CD8+ T Cells During BVDV Infection in vitro

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocyte (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the immunomodulatory effects of PD-1 pathway on major PBL subsets are unclear and their underlying molecular mechanisms need to be further studied. Therefore, in this study, we examined PD-1 expression in bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4+ and CD8+ T cells and expression of PD-1 downstream signaling molecules. The results showed that PD-1 expression was enhanced on CD4+ and CD8+ T cells, but not on CD21+ B cells after cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection in vitro and PD-1 blockade significantly reduced the apoptosis of CD4+ and CD8+ T cells after these two strains infection. Remarkably, PD-1 blockade significantly increased the proliferation of CD4+ and CD8+ T cells after CP BVDV infection, but only significantly increased the proliferation of CD4+ T cells after NCP BVDV infection. In addition, we confirmed that PD-1-mediated PI3K/Akt/mTOR, caspase 9/caspase 3 and ERK pathways are involved in regulating the apoptosis and proliferation of CD4+ and CD8+ T cells during BVDV infection in vitro. Notably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. Our findings provide a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocyte (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the immunomodulatory effects of PD-1 pathway on major PBL subsets are unclear and their underlying molecular mechanisms need to be further studied. Therefore, in this study, we examined PD-1 expression in bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4 + and CD8 + T cells and expression of PD-1 downstream signaling molecules. The results showed that PD-1 expression was enhanced on CD4 + and CD8 + T cells, but not on CD21 + B cells after cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection in vitro and PD-1 blockade significantly reduced the apoptosis of CD4 + and CD8 + T cells after these two strains infection. Remarkably, PD-1 blockade significantly increased the proliferation of CD4 + and CD8 + T cells after CP BVDV infection, but only significantly increased the proliferation of CD4 + T cells after NCP BVDV infection. In addition, we confirmed that PD-1-mediated PI3K/Akt/mTOR, caspase 9/caspase 3 and ERK pathways are involved in regulating the apoptosis and proliferation of CD4 + and CD8 + T cells during BVDV infection in vitro. Notably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. Our findings provide a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

INTRODUCTION
Bovine viral diarrhea virus (BVDV) is a significant cause of major economic losses in cattle worldwide and a potentiator for other diseases (1), including both cytopathic (CP) and non-cytopathic (NCP) biotypes (2). Acute BVDV infection is associated with immune dysfunction and can cause peripheral blood lymphopenia and apoptosis (3). There is huge difference in lymphopenia between strains (4-6). However, the molecular mechanism of the immune dysfunction is not entirely clear.
The programmed death-1 (PD-1) pathway plays a critical role in the regulation of immune response and the maintenance of lymphocyte homeostasis (7,8). Many viruses, such as human immunodeficiency virus (HIV) (9), hepatitis C virus (HCV) (10) and bovine leukemia virus (BLV) (11), can cause lymphocyte apoptosis, inhibit lymphocyte proliferation and induce functional exhaustion of lymphocytes via the PD-1 pathway. More notably, blocking PD-1 pathway improves T cell activation and proliferation and restores T cell function and antiviral immunity (12,13). To reveal the role of PD-1 in peripheral blood lymphopenia, apoptosis and immune dysfunction after acute BVDV infection, we have investigated the effect of BVDV infection on the expression of PD-1 and PD-L1 on peripheral blood mononuclear cells (PBMC) and analyzed the effect of PD-1 pathway on peripheral blood lymphocyte (PBL) immune functions by PD-1 blockade (14). We found that PD-1 and PD-L1 upregulation after CP and NCP BVDV infection was accompanied with decreased proliferation and increased apoptosis of PBL in vitro (14). Remarkably, PD-1 blockade inhibits PBL apoptosis and restores proliferation and anti-viral immune functions of PBL.
Nevertheless, the immunomodulatory effect of the PD-1 pathway on major PBL subsets, such as T helper cells, cytotoxic T cells and B cells in acute BVDV infection is still unclear. Moreover, the molecular mechanism underlying this immunomodulatory effect needs to be further studied. Therefore, in this study, we examined PD-1 expression on bovine PBL subsets after BVDV infection in vitro and analyzed the effects of PD-1 blockade on the apoptosis and proliferation of CD4 + and CD8 + T cells and the expression of PD-1 downstream signaling molecules. Our results provide a scientific basis for further exploration of the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Ethics Statement
This study was carried out in accordance with the principles of the Basel Declaration and recommendations followed by the guidelines set from College of Animal Science and Veterinary Medicine, HeiLongJiang Bayi Agricultural University. The protocol was approved by the Management Committee of the Experimental Animal Center of Heilongjiang Bayi Agricultural University.

Animals
Five healthy 1-year-old calvesfrom a Holstein dairy farm located in the city of Daqing in Heilongjiang Province, China were used for blood collection. The cattle were confirmed to be negative for BVDV antibody and BVDV as measured by antibody and antigen-capture ELISA test kits (IDEXX Laboratories, Westbrook, ME, USA) and negative for BLV, IBRV, and BIV infection as measured by PCR as previously described (15)(16)(17)(18).

Analysis of PD-1 Expression on PBL Subsets
The protein expressions of PD-1 on PBL subsets were measured by western blot analysis at 96 h post-infection (hpi) (14). Total protein was extracted, respectively from CD4 + T cells, CD8 + T cells, and CD21 + B cells with 100-150 µl RIPA buffer (Beytime, HangZhou, China) containing 15 mM PMSF (Beytime, HangZhou, China). Protein concentration was determined using the BCA Protein Assay Kit (Beytime, HangZhou, China) according to the manufacturer's instructions.

PD-1 Blockade Assay
To assess the effect of PD-1 pathway on apoptosis, viral replication and signaling molecules of BVDV-infected CD4 + and CD8 + T cells, a blocking assay was performed using anti-PD-1 mAb as described in the previous report (11,16). PBMC were infected with CP or NCP BVDV, respectively, and then cultured at 37 • C for 96 h in media supplemented with 10 ng/mL PMA and 500 ng/mL ionomycin in the presence of 10 µg/mL anti-PD-1 antibody (Abcam, Cambridge, UK) for detection of apoptosis, viral replication and signaling molecules. Anti-PD-1 antibody was added only once at the beginning. Frontiers in Immunology | www.frontiersin.org

Cell Apoptosis Measurement Using Flow Cytometry
The CD4 + and CD8 + T cells were selected, respectively, from the PBMC using magnetic cell separation technique. These cells were stained using Annexin-V-FITC apoptosis Kit with propidium iodide for 15 min at room temperature in the dark according to the manufacturer's instructions (Beyotime, Shanghae, China) and analyzed immediately on a CytoFLEX flow cytometer (Beckman Coulter, USA).

Cell Proliferation Assay
The CD4 + and CD8 + T cells were individually co-cultured with CD14 + monocytes and infected with CP or NCP BVDV, respectively. The cells were cultured at 37 • C in media containing 10 ng/mL PMA and 500 ng/mL ionomycin in the presence of 10 µg/mL anti-PD-1 antibody for 7 days in an incubator supplemented with 5% CO 2 . Anti-PD-1 antibody was added only once at the beginning. The infected cells without antibody were used as the controls. Cell proliferation was detected using cell counting kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. The density of cells in each well was measured according to the absorbance at 450 nm every 24 h.

Effect of PD-1 Blockade on Virus Replication
To evaluate the effects of PD-1 blockade on the time course of virus replication in CD4 + and CD8 + T cells, nucleotide Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org sequences of 5 ′ non-coding region (NCR) of BVDV was amplified and cloned into a pMD18-T vector for the establishment of qRT-PCR as standard DNA (11). Primers were used as follow, 5 ′ -GAG TAC AGG GTA GTC GTC AG-3 ′ and 5 ′ -CTC TGC AGC ACC CTA TCA GG-3 ′ for 5 ′ NCR, and 5 ′ -CGC ACC ACT GGC ATT GTC AT-3 ′ and 5 ′ -TCC AAG GCG ACG TAG CAG AG-3 ′ for β-actin. The recombinant plasmid pMD18-T/5 ′ NCR was serially diluted 10-fold in TE buffer, pH 8.0, from 10 −1 to 10 −7 . Each dilution of them was tested in triplicate and used as an amplification template to construct standard curves to determine the copy number of the BVDV 5 ′ NCR gene. Quantitative real-time PCR was performed in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using SYBR Premix Ex Taq II (TaKaRa Biotechnology, Dalian, China) following the manufacturer's instructions. Each cDNA sample from PBMCs suspension was tested in triplicate; the cycling conditions consist of initial template denaturing at 95 • C for 30 s, followed by amplification of template for 45 cycles of 95 • C for 5 s, 60 • C for 30 s and 72 • C for 30 s. A final melting curve analysis was performed from 65 to 95 • C at a rate of 0.1 • C/s (continuous acquisition), with a final cooling to 40 • C over 10 s.
The results are shown as the mean copy number per milliliter of cell suspension.

Statistical Analysis
All data were expressed as mean ± SD and analyzed using student's unpaired t-test, one-way ANOVA and two-way ANOVA using GraphPad Prism version 6.0 (GraphPad Software). A p < 0.05 indicated a statistically significant difference. All samples were assayed in triplicate.

Expression of PD-1 on PBL Subsets After BVDV Infection
Our previous report (14) has confirmed that PD-1 expression was significantly upregulated on both the CP BVDV and the NCP BVDV infected PBL compared with uninfected PBL. To study the protein expressions of PD-1 on PBL subsets after infection with BVDV, western blot analysis was performed in this study. The results showed that there was a significant increase in PD-1 expression on CD4 + (CP BVDV, p < 0.01, Figure 1A; NCP BVDV, p < 0.001, Figure 2A) and CD8 + T cells (CP BVDV, p < 0.001, Figure 1B; NCP BVDV, p < 0.05, Figure 2B) after both the CP BVDV and the NCP BVDV infection. However, there was no significant increase in PD-1 expression on CD21 + B cells (Figures 1C, 2C). Original data is shown in Figures S20-S22 in Supplementary Material.

Detection of Apoptosis in CD4 + and CD8 + T Cells
To assess the effect of PD-1 pathway on the apoptosis of BVDV-infected CD4 + and CD8 + T cells, we investigated the cell apoptosis by flow cytometric analysis. The results showed that the proportion of apoptotic cells was significantly reduced in the infected CD4 + (CP BVDV, p < 0.05, Figure 3A; NCP BVDV, p < 0.05, Figure 3C) and CD8 + T cells (CP BVDV, p < 0.01, Figure 3B; NCP BVDV, p < 0.05, Figure 3D) by PD-1 blockade.

Measurement of CD4 + and CD8 + T Cells Proliferation
To investigate the effect of PD-1 pathway on the proliferation of BVDV-infected CD4 + and CD8 + T cells, we detected the cell proliferation using cell counting kit-8. PD-1 blockade significantly restored proliferation of the CP BVDV infected CD4 + and CD8 + T cells from 72 to 144 hpi (Figures 4A,B) and the proliferation of the NCP BVDV infected CD4 + T cells at 72 and 96 hpi (p < 0.05, Figure 4C), but not NCP BVDV infected CD8 + T cells ( Figure 4D).

Viral Replication
To assess the effects of PD-1 blockade on the time course of virus replication, we investigated the BVDV load in the infected CD4 + and CD8 + T cells by quantitative real-time PCR from 24 to 96 hpi. After treatment with an anti-PD-1 antibody, the replication of the CP BVDV and NCP BVDV were significantly inhibited at 72 and 96 hpi in infected CD4 + (CP BVDV, Figure 5A; NCP BVDV, Figure 5C) and CD8 + T cells (CP BVDV, Figure 5B; NCP BVDV, Figure 5D). Moreover, observation using CLSM (confocal laser scanning microscopy) intuitively showed that most CD4 + and CD8 + T cells were infected by virus at 96 hpi (CP BVDV, Figures 6A,B; NCP BVDV, Figures 6C,D).

Effects of PD-1 Blockade on the Expression of Downstream Signaling Molecules
To determine the effects of PD-1 blockade on the expression and phosphorylation of downstream signaling molecules, western blot analysis was performed. In the CP BVDV infected CD4 + and CD8 + T cells, the expression levels of p-PI3K, p-Akt, p-mTOR and p-ERK were significantly upregulated by PD-1 blockade (CD4 + T cells, Figures 7A,B,E-G; CD8 + T cells,  , cleaved-caspase 9 (C), cleaved-caspase 3 (D), p-mTOR (E), p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. ***p < 0.001, **p < 0.01, *p < 0.05. Data are presented as mean ± SD (n = 5 per group). Figures 8A,B,E-G). Meanwhile, the expression levels of cleavedcaspase 9 and cleaved-caspase 3 were significantly downregulated (CD4 + T cells, Figures 7C,D; CD8 + T cells, Figures 8C,D). Furthermore, in the NCP BVDV infected CD4 + and CD8 + T cells, we observed a significant increase in p-PI3K and p-Akt (CD4 + T cells, Figures 9A,B,G; CD8 + T cells, Figures 10A,B,G), and a significant decrease in cleaved-caspase 9 and cleavedcaspase 3 by PD-1 blockade (CD4 + T cells, Figures 9C,D; CD8 + T cells, Figures 10C,D). Remarkably, the expression level of p-mTOR was significantly upregulated in the NCP BVDV infected CD4 + T cells (p < 0.01, Figure 9E) but not significantly changed in the NCP BVDV infected CD8 + T cells (Figure 10E) by PD-1 blockade. In addition, PD-1 blockade had no significant effect on p-ERK expression in the NCP BVDV infected CD4 + and CD8 + T cells (Figures 9F, 10F). Original data is shown in Figures S1-S19 in Supplementary Material. (19,20). Regulation of T cell activation requires not only the recognition of antigen , cleaved-caspase 9 (C), cleaved-caspase 3 (D), p-mTOR (E), p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. ***p < 0.001, **p < 0.01. Data are presented as mean ± SD (n = 5 per group). by the antigen specific TCR but also co-signaling molecules categorized as co-stimulatory and co-inhibitory molecules (21). PD-1, as a co-inhibitory molecule, inhibits TCR-mediated activation and proliferation, and induces lymphocyte apoptosis and immune dysfunction by binding to its ligand such as PD-L1 (22). In a previous study we reported that PD-1 plays a vital role in peripheral blood lymphopenia and apoptosis caused by acute BVDV infection (14). However, it is unclear what the immunomodulatory effect of the PD-1 pathway is on major PBL subsets. Moreover, the molecular mechanism underlying this effect needs to be further studied. It has been shown that PD-1 was upregulated on CD8 + and CD4 + T cells and that its expression increased apoptosis of HIV-specific CD8 + T cells (23) and showed an inverse correlation with CD4 + T cell count (9). Studies on HBV infection demonstrated that blocking PD-1-mediated pathway in vitro reduced HBV-specific CD8 + T cell apoptosis and IL-10 production (24). In this study, we demonstrated that an increase in PD-1 expression in CD4 + and in CD8 + T subgroup but not in CD21 + B cells when the cells were infected with BVDV. Meanwhile, PD-1 blockade significantly reduced apoptosis and viral load in CD4 + and CD8 + T cells after BVDV infection. Our results suggest that the PD-1 pathway regulates apoptosis of CD4 + and FIGURE 9 | Effect of PD-1 blockade on downstream signaling molecules in NCP BVDV-infected CD4 + T cells. Shown are the results of densitometric analyses of the levels of p-PI3K (A), p-Akt (B), cleaved-caspase 9 (C), cleaved-caspase 3 (D), p-mTOR (E), p-ERK (F) in bar graph format as well as the representative results (G) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, cleaved-caspase 9, cleaved-caspase 3, mTOR, p-mTOR, ERK, p-ERK, and β-actin. The infected CD4 + and CD8 + T cells without antibody were used as control groups. N.S., not significant, ***p < 0.001, **p < 0.01. Data are presented as mean ± SD (n = 5 per group).

CD8 + T cells during the CP BVDV and the NCP BVDV infection in vitro.
PD-1 also inhibits BCR-mediated B cell activation and proliferation (25), which leads to the prevention of autoimmune diseases. However, very few studies have been done on PD-1 regulating B cell survival and functions during viral infection. It has been shown that PD-1 expression was increased on CD21 + CD27 + resting memory B cells in HIV-1-infected patients, which might contribute to a decreased ratio of the resting memory B cells during HIV-1 infection (26). Notably, B cells are also important target of BVDV, and their apoptotic effect on B cells depends on the strain (27). We focused on lymphocyte subsets with high expression of PD-1 in this study. Infection of both CP BVDV and NCP BVDV increased PD-1 expression in CD21 + B cells, but these increases were not significant. Infection of both CP BVDV and NCP BVDV may affect PD-1 expression in CD21 + subpopulations such as CD21 + CD27 + resting memory B cells. The relationship between B cell apoptosis and PD-1 need to be further explored.
phosphorylation of ZAP-70 and initiation of downstream signal pathways such as PI3K/Akt and ERK pathways (29). The PI3K/Akt/mTOR pathway can regulate cell proliferation and apoptosis. Abnormally elevated mTOR expression promotes tumor proliferation and metastasis in many human malignancies (30). The mTOR pathway also plays an important role in innate and acquired immunity. Activated PI3K/Akt/mTOR pathway can improve T lymphocyte metabolism, nutrient uptake and energy production, regulate cell cycle and apoptosis, and affect T lymphocyte activation and immune function (31)(32)(33)(34). Blocking the interaction between PD-1 and PD-L1 can reactivate PI3K/Akt/mTOR pathway and restore immune function of exhausted CD8 + T cells (35). In addition, the activation of the Ras/MEK/ERK pathway can promote cell protein synthesis and proliferation. PD-1 blockade restored T lymphocyte proliferation and improve its antiviral function by reactivating the Ras/MEK/ERK pathway (29). Caspase 9 is a key pro-apoptotic signaling molecule downstream of the PI3K/Akt pathway and an essential initiator of the mitochondrial apoptosis pathway (36). The PI3K/Akt pathway plays an anti-apoptotic role through inhibiting the caspase 9 signaling cascade (37). In BVDV infected cattle, the expression of caspase 9 and caspase 3 is involved in lymphoid tissues lesions, but whether it directly induces apoptosis in infected cells or whether it is related to the PD-1 pathway remains unclear (38). In our study, by measuring the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, cleaved-caspase 9, and cleaved-caspase 3, we confirmed that CP BVDV infection can inhibit the proliferation of CD4 + and CD8 + T cells and induce apoptosis via PD-1-mediated repression of PI3K/Akt/mTOR and ERK pathways and activation of the caspase 9/caspase 3 pathway. NCP BVDV infection inhibits the proliferation of CD4 + T cells by suppressing of PI3K/Akt/mTOR pathway, and induces the apoptosis of CD4 + and CD8 + T cells by activating the caspase 9/caspase 3 pathway.
Overexpression of PD-1 can inhibit proliferation of virusspecific CD4 + and CD8 + T cells (9,39), and PD-1 blockade can restore proliferation and anti-viral immune functions of T cells (24,40). However, PD-1 blockade in NCP BVDV infected cells did not significantly affect proliferation and p-mTOR and p-ERK levels of CD8 + T cells. Meanwhile, PD-1 blockade had a more significant effect on the decrease of viral load in CP BVDV infected CD8 + T cells than in NCP BVDV infected CD8 + T cells. It is known that activation and proliferation of T cells are negatively regulated by many co-inhibitory molecules, such as Tim-3 (41) and CTLA-4 (42). Tim-3 expression was upregulated on HIV-1 infected CD8 + T cells and associated with the inhibition of T cell proliferation and the impairment of ERK signaling (43). Blocking the co-inhibitory molecule Tim-3 with antibodies can enhance the proliferation of HIV-specific CD8 + T cells (44). CTLA-4 inhibits T cell proliferation and IL-2 production by suppressing TCR/CD28-induced ERK and JNK activation (45). In addition, the combined blockade of PD-1/CTLA-4 can synergistically reverse exhaustion of HCV-specific CD8 + T cells (46). Notably, both HCV and BVDV belong to the Flaviviridae family. In addition, previous study on PD-1 blockade has confirmed the synergistic effect of PD-1 and other co-inhibitory molecules on T cells dysfunction during HIV (44) and HCV (45) infections. However, it is unclear whether PD-1 cooperates with other co-inhibitory molecules to regulate T cells dysfunction during BVDV infection, especially during NCP BVDV infection. Consistently, the blocking assays in this study also revealed that PD-1 is associated with T cells dysfunction, but the possible role of other co-inhibitory molecules needs to better evaluate by more optimized experimental designs, such as the combination of blocking assay and silencing assay.
In summary, we have demonstrated enhancement of PD-1 expression on BVDV-infected CD4 + and CD8 + T cells, but not B cells, following infection with CP BVDV and NCP BVDV in vitro. PD-1 blockade significantly reduced apoptosis of T cells and increased proliferation of CD4 + and CD8 + T cells after CP BVDV infection and increased proliferation of CD4 + T cells after NCP BVDV infection. We also reported the involvement of PI3K/Akt/mTOR, ERK, and caspase 9/caspase 3 pathways in CP BVDV induction of apoptosis and inhibition of CD4 + and CD8 + T cell proliferation. These pathways were shown to have a similar effect on CD4 + T cells following NCP BVDV infection. Remarkably, ERK is involved in the regulation mechanism PD-1 mediated only when the cells are infected with CP BVDV. In addition, a weakness of this paper is that bovine WBC were not taken during the experiment. The situation in vivo remains to be further studied and confirmed.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The protocol was approved by the Management Committee of the Experimental Animal Center of Heilongjiang Bayi Agricultural University.

AUTHOR CONTRIBUTIONS
YL: data collection, data analysis and interpretation, and drafting the article. WH and CW: data collection and data analysis and interpretation. SLia and LW: data analysis and interpretation and drafting the article. YL and BX: critical revision of the article. SLiu and NC: data analysis and interpretation and critical revision of the article. SY: conception or design of the work and critical revision of the article. ZZ: conception or design of the work, data analysis and interpretation, drafting the article, and critical revision of the article.