Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFβ1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFβ1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFβ1 mRNA expression and protein translation. Transfection of TGFβAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFβ1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNβ, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFβAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2'-5'-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFβAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFβ expression. These findings also suggest TGFβ as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.

Keywords: Antisense; Gene knockdown; Innate immunity; Porcine reproductive and respiratory syndrome virus; Transforming growth factor beta.

Fig. 1

Optimization and validation of knockdown of porcine TGFβ1 mRNA expression and protein translation by AS ODNs. A) MDMs under bright-field microscopy. B) MDMs uptake of fluorescent-labeled siRNA under immunofluorescent microscopy. C) MDMs uptake of fluorescent-labeled siRNA complexed with different concentrations of transfection reagent and transfection period. D) Effect of TGFβ1 antisense (AS1-4), sense (S1-4) and scramble (Scr1-4) phosphorothioate-modified ODNs on the expression of TGFβ1 mRNA in MDMs stimulated with a mixture of poly I:C and LPS. Band intensities (Additional file 1) indicate the quality of TGFβ1 knockdown. E) Optimization of TGFβAS1 concentration for TGFβ1 mRNA knockdown on MDMs transfected with TGFβAS1 (0.5, 1, or 2 µM) and stimulated with a mixture of poly I:C and LPS. Band intensities (Additional file 2) indicate the quality of TGFβ1 knockdown. F) TGFβ1 protein levels in MDMs transfected with TGFβAS1 (2 µM) and stimulated with a mixture of poly I:C and LPS. In all figures, error bars indicate the standard deviation (SD). Mean differences of TGFβ1 gene expression or protein translation among groups were tested by one-way ANOVA, followed by Tukey HSD test. Mean differences of percentages of fluoresced cells among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD test. Different letters indicate significant differences. P < 0.05 was set as a statistically significant level

Fig. 2

Heat map illustrating expression levels of immune-related genes in MDMs transfected with either TGFβAS1 (I) or Scr1 (II), or otherwise treated with transfection media (III) alone prior to stimulation with a mixture of poly I:C and LPS. Untransfected MDMs stimulated with a mixture of poly I:C and LPS served as positive control (IV). Data were normalized to the geometric average of RPL32 and YWHAZ relative to untransfected/unstimulated MDMs. Data are presented in log 2 scale of “fold” according to 2^(-ΔΔC_T) method

Fig. 3

Heat map illustrating effects of TGFβAS1 on immune-related gene expressions in PRRSV-2-inoculated MDMs. MDMs were transfected with TGFβAS1, then inoculated with either cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS. MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control. MDMs treated with transfection media (Tr. media) and inoculated with cPRRSV-2 or HP-PRRSV-2, then stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated/Tr. media control. MDMs inoculated with mock Ag and stimulated with a mixture of poly I:C and LPS served as mock control. Untreated MDMs stimulated with a mixture of poly I:C and LPS served as positive control (Pos Ctrl). I = cPRRSV-2; II = HP-PRRSV-2; III = Tr. media + cPRRSV-2; IV = Tr. media + HP-PRRSV-2; V = TGFβAS1 + cPRRSV-2; VI = TGFβAS1 + HP-PRRSV-2; VII = Mock Ag; VIII = Pos Ctrl. Data were normalized to the geometric average of RPL32 and YWHAZ relative to untransfected/unstimulated MDMs. Data are presented in log 2 scale of “fold” according to 2^(-ΔΔC_T) method

Fig. 4

Effect of TGFβAS1 on TGFβ1 protein translation in PRRSV-2-inoculated MDMs. MDMs were transfected with TGFβAS1, then inoculated with either cPRRSV-2 or HP-PRRSV-2, and stimulated with a mixture of poly I:C and LPS. MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control. MDMs treated with transfection media (Tr. media) and inoculated with cPRRSV-2 or HP-PRRSV-2, then stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated/Tr. media control. MDMs inoculated with mock Ag and stimulated with a mixture of poly I:C and LPS served as mock control. Untreated MDMs receiving culture media in the presence or absence of a mixture of poly I:C and LPS served as positive and negative controls, respectively. Cell culture supernatants were collected for ELISA. Error bars indicate the SD. Mean differences of TGFβ1 protein translation among groups were tested by one-way ANOVA, followed by Tukey HSD test. Different letters indicate significant differences. P < 0.05 was set as a statistically significant level

Fig. 5

Effect of TGFβ knockdown on PRRSV copy numbers in PRRSV-2-inoculated MDMs. MDMs were transfected with TGFβAS1, then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h). MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control. MDMs transfected with Scr1, then inoculated with either cPRRSV-2 or HP-PRRSV-2, and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated/Scr1 control. MDMs treated with transfection media (Tr. media), then inoculated with cPRRSV-2 or HP-PRRSV-2, and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated/Tr. media control. MDMs receiving mock Ag plus a mixture of poly I:C and LPS served as uninoculated control. Cell culture supernatants were collected for real-time PCR. The C_T values were obtained and PRRSV-2 ORF7 RNA copy numbers were calculated based on the C_T standard curve generated from 10¹-10⁸ copies of recombinant PRRSV-2 ORF7 plasmids. Data were presented in log 10 scale of copy number/ml. Error bars indicate the SD. Mean differences of PRRSV-2 ORF7 RNA copy numbers among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD. Different superscript letters indicate significant difference. P < 0.05 was set as a statistically significant level

Fig. 6

Effects of rIFNα on PRRSV-2 ORF7 RNA copy numbers in PRRSV-2-inoculated MDMs. MDMs were treated with rIFNα (10, 1 and 0.1 ng/ml final), then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h). MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control. MDMs receiving mock Ag plus a mixture of poly I:C and LPS served as uninoculated control. Cell culture supernatants were collected for real-time PCR. The C_T values were obtained and PRRSV-2 ORF7 RNA copy numbers were calculated based on the C_T standard curve generated from 10¹-10⁸ copies of recombinant PRRSV-2 ORF7 plasmids. Data were presented in log 10 scale of copy number/mL. Error bars indicate the SD. Mean differences of PRRSV-2 ORF7 RNA copy numbers among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD. Different superscript letters indicate significant differences. P < 0.05 was set as a statistically significant level

Fig. 7

Effects of rTGFβ1 and rIFNα on PRRSV-2 ORF7 RNA copy numbers in PRRSV-2-inoculated MDMs. MDMs were treated with rTGFβ1 (10 ng/ml final), followed by rIFNα (10 ng/ml final), then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h). MDMs inoculated with cPRRSV-2 or HP-PRRSV-2 and stimulated with a mixture of poly I:C and LPS served as PRRSV-2-inoculated control. MDMs treated with rTGFβ1, then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h) served as rTGFβ1-treated/PRRSV-2-inoculated control. MDMs treated with rIFNα, then inoculated with either cPRRSV-2 or HP-PRRSV-2 (0 h), and stimulated with a mixture of poly I:C and LPS (48 h) served as rIFNα -treated/PRRSV-2-inoculated control. MDMs receiving mock Ag plus a mixture of poly I:C and LPS served as uninoculated control. Cell culture supernatants were collected for real-time PCR. The C_T values were obtained and PRRSV-2 ORF7 RNA copy numbers were calculated based on the C_T standard curve generated from 10¹-10⁸ copies of recombinant PRRSV-2 ORF7 plasmids. Data were presented in log 10 scale of copy number/mL. Error bars indicate the SD. Mean differences of PRRSV-2 ORF7 RNA copy numbers among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD. Different superscript letters indicate significant differences. P < 0.05 was set as a statistically significant level