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The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation.

Fan B, Liu X, Bai J, Li Y, Zhang Q, Jiang P - PLoS ONE (2015)

Bottom Line: Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression.By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation.The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15-21, 42-48 and 88-94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.

No MeSH data available.


Related in: MedlinePlus

Induction of Tregs from lymphocytes co-cultured with PRRSV-infected MoDCs.(A) Representative flow cytometry profile of lymphocytes following 3 days co-culture of PBMCs with PRRSV-infected MoDCs (upper layer), and only culture with PRRSV-infected PBMCs (lower layer). (B) Percentage of Foxp3+ cells in the gated CD4+CD25+ subpopulations of PBMCs co-cultured with PRRSV-infected MoDC. (C) Percentage of Foxp3+ cells in the gated CD4+ CD25+ subpopulations of PRRSV-infected PBMCs alone. (D) Sorted CD4+CD25high cells. (E) Percentage suppression at the indicated Tregs: peripheral lymphocyte ratios. The lymphocytes exposed to Marc-145 lysate-treated MoDCs were used as negative control. The percentage of suppression was calculated as follows: % suppression = 100 × [1 − (% proliferation w/PRRSV/ % proliferation w/mock)] [35]. Data from three independent experiments. All data analysis was done using one-way ANOVA and significant differences are shown (*P<0.05 and **P< 0.01).
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pone.0138772.g001: Induction of Tregs from lymphocytes co-cultured with PRRSV-infected MoDCs.(A) Representative flow cytometry profile of lymphocytes following 3 days co-culture of PBMCs with PRRSV-infected MoDCs (upper layer), and only culture with PRRSV-infected PBMCs (lower layer). (B) Percentage of Foxp3+ cells in the gated CD4+CD25+ subpopulations of PBMCs co-cultured with PRRSV-infected MoDC. (C) Percentage of Foxp3+ cells in the gated CD4+ CD25+ subpopulations of PRRSV-infected PBMCs alone. (D) Sorted CD4+CD25high cells. (E) Percentage suppression at the indicated Tregs: peripheral lymphocyte ratios. The lymphocytes exposed to Marc-145 lysate-treated MoDCs were used as negative control. The percentage of suppression was calculated as follows: % suppression = 100 × [1 − (% proliferation w/PRRSV/ % proliferation w/mock)] [35]. Data from three independent experiments. All data analysis was done using one-way ANOVA and significant differences are shown (*P<0.05 and **P< 0.01).

Mentions: To evaluate the role of HP-PRRSV in induction of Tregs, porcine MoDCs were generated from PBMCs using rpGM-CSF and rpIL-4. The porcine MoDCs exhibited significant enhanced expressions of CD80, CD86 and MHC class II molecules on the cellular surface, compared with the freshly isolated lymphocytes (S2 Fig). This result indicated that the MoDCs were generated successfully. In addition, the HP-PRRSV strain could successfully infect MoDCs as previous reports (S3 Fig) [33, 36]. As shown in Fig 1, The CD4+ CD25+ Foxp3+ expression was determined on autologous PBMCs co-cultured with HP-PRRSV-infected MoDCs using flow cytometry. The percentage of CD4+CD25+Foxp3+ Tregs among HP-PRRSV-infected MoDCs was significantly higher than that co-cultured with mock-infected MoDCs (P<0.01) (Fig 1A and 1B). The percentage of Tregs was not significantly increased in HP-PRRSV-infected PBMCs alone, compared with that treated with Marc-145 cell lysates (M-Lysates) (P>0.05) (Fig 1A and 1C).


The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation.

Fan B, Liu X, Bai J, Li Y, Zhang Q, Jiang P - PLoS ONE (2015)

Induction of Tregs from lymphocytes co-cultured with PRRSV-infected MoDCs.(A) Representative flow cytometry profile of lymphocytes following 3 days co-culture of PBMCs with PRRSV-infected MoDCs (upper layer), and only culture with PRRSV-infected PBMCs (lower layer). (B) Percentage of Foxp3+ cells in the gated CD4+CD25+ subpopulations of PBMCs co-cultured with PRRSV-infected MoDC. (C) Percentage of Foxp3+ cells in the gated CD4+ CD25+ subpopulations of PRRSV-infected PBMCs alone. (D) Sorted CD4+CD25high cells. (E) Percentage suppression at the indicated Tregs: peripheral lymphocyte ratios. The lymphocytes exposed to Marc-145 lysate-treated MoDCs were used as negative control. The percentage of suppression was calculated as follows: % suppression = 100 × [1 − (% proliferation w/PRRSV/ % proliferation w/mock)] [35]. Data from three independent experiments. All data analysis was done using one-way ANOVA and significant differences are shown (*P<0.05 and **P< 0.01).
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Related In: Results  -  Collection

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pone.0138772.g001: Induction of Tregs from lymphocytes co-cultured with PRRSV-infected MoDCs.(A) Representative flow cytometry profile of lymphocytes following 3 days co-culture of PBMCs with PRRSV-infected MoDCs (upper layer), and only culture with PRRSV-infected PBMCs (lower layer). (B) Percentage of Foxp3+ cells in the gated CD4+CD25+ subpopulations of PBMCs co-cultured with PRRSV-infected MoDC. (C) Percentage of Foxp3+ cells in the gated CD4+ CD25+ subpopulations of PRRSV-infected PBMCs alone. (D) Sorted CD4+CD25high cells. (E) Percentage suppression at the indicated Tregs: peripheral lymphocyte ratios. The lymphocytes exposed to Marc-145 lysate-treated MoDCs were used as negative control. The percentage of suppression was calculated as follows: % suppression = 100 × [1 − (% proliferation w/PRRSV/ % proliferation w/mock)] [35]. Data from three independent experiments. All data analysis was done using one-way ANOVA and significant differences are shown (*P<0.05 and **P< 0.01).
Mentions: To evaluate the role of HP-PRRSV in induction of Tregs, porcine MoDCs were generated from PBMCs using rpGM-CSF and rpIL-4. The porcine MoDCs exhibited significant enhanced expressions of CD80, CD86 and MHC class II molecules on the cellular surface, compared with the freshly isolated lymphocytes (S2 Fig). This result indicated that the MoDCs were generated successfully. In addition, the HP-PRRSV strain could successfully infect MoDCs as previous reports (S3 Fig) [33, 36]. As shown in Fig 1, The CD4+ CD25+ Foxp3+ expression was determined on autologous PBMCs co-cultured with HP-PRRSV-infected MoDCs using flow cytometry. The percentage of CD4+CD25+Foxp3+ Tregs among HP-PRRSV-infected MoDCs was significantly higher than that co-cultured with mock-infected MoDCs (P<0.01) (Fig 1A and 1B). The percentage of Tregs was not significantly increased in HP-PRRSV-infected PBMCs alone, compared with that treated with Marc-145 cell lysates (M-Lysates) (P>0.05) (Fig 1A and 1C).

Bottom Line: Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression.By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation.The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15-21, 42-48 and 88-94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.

No MeSH data available.


Related in: MedlinePlus