Type I and II interferons, transcription factors and major histocompatibility complexes were enhanced by knocking down the PRRSV-induced transforming growth factor beta in monocytes co-cultured with peripheral blood lymphocytes

The innate and adaptive immune responses elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection are known to be poor. This study investigates the impact of PRRSV-induced transforming growth factor beta 1 (TGFβ1) on the expressions of type I and II interferons (IFNs), transcription factors, major histocompatibility complexes (MHC), anti-inflammatory and pro-inflammatory cytokines in PRRSV-infected co-cultures of monocytes and peripheral blood lymphocytes (PBL). Phosphorothioate-modified antisense oligodeoxynucleotide (AS ODN) specific to the AUG region of porcine TGFβ1 mRNA was synthesized and successfully knocked down TGFβ1 mRNA expression and protein translation. Monocytes transfected with TGFβAS1 ODN, then simultaneously co-cultured with PBL and inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) showed a significant reduction in TGFβ1 mRNA expression and a significant increase in the mRNA expressions of IFNα, IFNγ, MHC-I, MHC-II, signal transducer and activator of transcription 1 (STAT1), and STAT2. Additionally, transfection of TGFβAS1 ODN in the monocyte and PBL co-culture inoculated with cPRRSV-2 significantly increased the mRNA expression of interleukin-12p40 (IL-12p40). PRRSV-2 RNA copy numbers were significantly reduced in monocytes and PBL co-culture transfected with TGFβAS1 ODN compared to the untransfected control. The yields of PRRSV-2 RNA copy numbers in PRRSV-2-inoculated monocytes and PBL co-culture were sustained and reduced by porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα), respectively. These findings highlight the strategy employed by PRRSV to suppress the innate immune response through the induction of TGFβ expression. The inclusion of TGFβ as a parameter for future PRRSV vaccine and vaccine adjuvant candidates is recommended.

PRRSV enhances IL-10 expression (27,28).IL-10 upregulation, in conjunction with limited expressions of interferon-regulated genes, contributes to the downregulation of pro-inflammatory innate immune defense mechanisms e.g., antiviral and phagocytic activities, antigen presentation, pro-inflammatory cytokines and immune-related marker expressions in infected myeloid APCs.The weak and delayed inductions of adaptive cytotoxic T cell (29) and T helper 1 (Th1) cell responses, as well as the promotion of regulatory Tregs differentiation, further facilitate the survival of PRRSV and the development of clinical manifestations (30)(31)(32).
Transforming growth factor beta (TGFb) has three isoforms in mammals, with TGFb1 being the most abundant isoform and responsible for a wide range of specific responses (33).In pigs, the impact of PRRSV-induced TGFb1 overexpression on immune protection against PRRSV has not been investigated to date.TGFb is up-regulated by PRRSV in infected myeloid APCs (34), cocultivated peripheral blood mononuclear cells (PBMCs) (27), lungs and lymphoid tissues of pigs (35).According to the previous study, TGFb can enhance the viability of PRRSV-infected cells, thereby contributing to increased PRRSV survival (36).Limited existing reports on the immunoregulatory activities of TGFb in pigs are available.In porcine monocyte-derived macrophages (MDMs), TGFb has been reported to down-regulate the expressions of CD14, IL-6, MHCII, and tumor necrosis factor alpha (TNFa) (37).In mice, TGFb has been found to down-regulate CD14 expression in lipopolysaccharide (LPS)-stimulated macrophages, leading to the suppression of the MyD88-dependent signaling pathway (38,39).Moreover, TGFb has been shown to suppress MHCII, IL-12p40, and CD40 expressions in murine macrophages (38), Th1 cell differentiation, Th1-mediated inflammatory response, and the expressions of IFNg, IL-2, and IL-4 (40) as well as the activation of macrophages, dendritic cells (DCs), and natural killer cells (38).In contrast, TGFb promotes Tregs differentiation through the upregulation of Smad3 and Foxp3 expressions (41).
This study aims to investigate the effects of PRRSV-induced TGFb on the expressions of immune-related genes in PRRSVinoculated monocytes and PBL co-culture.Monocytes were transfected with phosphorothioate-modified antisense (AS) oligodeoxynucleotide (ODN) targeting the AUG region of porcine TGFb1 mRNA to knockdown its expression.The present study reveals that TGFb exerts a regulatory role by down-regulating gene expressions of type I and II IFNs, MHCs, and STATs in PRRSVinoculated monocytes and PBL co-culture.These findings provide valuable insights into potential targets and strategies for augmenting the innate and cell-mediated immune (CMI) responses to PRRSV vaccines and vaccine adjuvants.

Pigs
Eight 24-week-old PRRSV-seronegative crossbred pigs (large white/landrace x duroc) were the source of peripheral blood mononuclear cells (PBMCs) to obtain monocytes and PBLs.They were housed at the swine research farm, faculty of animal science and technology, Maejo University.
Total RNA was isolated using PureLink ™ RNA mini kit (Invitrogen, USA).The quality and quantity of RNA were evaluated by the OneDrop TOUCH Pro/Lite Micro-Volume Spectrophotometer (Biometrics Technologies Inc. USA).All RNA samples had A260/230 and A260/280 between 1.8-2.2 and 2.0-2.2,respectively.The integrity of RNA was determined by denaturing agarose gel electrophoresis followed by ethidium bromide staining.cDNA was carried out using the ReverTra Ace ® qPCR RT Kit (Toyobo, Japan).The reaction used 1,000 ng of pooled total RNA as a template, and a mixture of random hexamers and oligo-dT as primers.cDNAs were stored at -20°C until real-time PCR.
Transfection was carried out following the guidelines of Lipofectamine ™ RNAiMax (Invitrogen, CA) with the recommended small interfering RNA (siRNA, BLOCK-iT ™ Alexa Fluor ® Red Fluorescent control, Invitrogen).Mixtures of 0.75, 1.5 and 3% Lipofectamine ™ RNAiMax in Opti-MEM ® I (v/v) and 2 µM siRNA suspended in Opti-MEM ® I were incubated at room temperature (RT) for 30 min.Twenty µl of each mixture was added to the wells containing monocytes and gently mixed by rocking for 5 min.Monocyte uptake of fluorescent-labeled siRNA (Figures 1A, B) was observed under an immunofluorescent microscope (Nikon Eclipse Ti, Japan) every 2 h for 10 h and finally at 24 h.Frequencies of immunofluorescent-positive cells were identified using automatic measurement for cell counting (NIS-elements software ver.3.22, Nikon, Japan).Cell viability was determined by trypan blue staining.Optimal concentration of transfection reagent and optimal transfection period were determined.

TGFb1 knockdown in monocytes then co-cultured with peripheral blood lymphocytes prior to the evaluation of immune-related gene expressions
Monocytes were prepared as described above while PBLs were prepared as previously described (44).Monocytes were washed with 100 µl pre-warmed (37°C) Opti-MEM ® I and transfected with 40 µl TGFbAS1 mixture for 4 h (37°C, humidified 5% CO 2 ).The transfection condition was based on the results of optimization of transfection reagent concentration and transfection period (Figure 1C, Additional File 2).The TGFbAS1 mixture was removed, and adherent TGFbAS1-transfected monocytes were washed.Then, 200 µl of PBL (2 x 10 6 cells) in RPMI ++ from the same animal were added to the TGFbAS1-transfected monocyte for co-culture.The ratio of PBL added to monocytes resembled the ratio of lymphocytes to monocytes in the peripheral blood of pigs of the same age, approximately 10:1 (47).Finally, 50 µl of inducer, either ConA (10 mg/ ml final conc.) or PMAi (7 and 430 ng/ml final conc.) was added to the wells and incubated for an additional 12 h (37°C, humidified 5% CO 2 ).
For the determination of mRNA expression levels of TGFb1 and other immune-related genes (Additional File 1), 200 ng of total RNA was used as template for cDNA synthesis as described above.The threshold cycles (C T ) of all genes were used for the calculation of gene expression by the 2^(-DDC T ) method.The expressions of TGFb1 and other immune-related genes were normalized to the geometric average of RPL32 (ribosomal protein L32) and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta) and calibrated to that in the negative control.The expression levels of all immune-related genes were transformed into log2 scale.

Evaluation of TGFb1 knockdown effects on immune-related gene expressions in monocytes co-cultured with PBL and inoculated with cPRRSV-2 and HP-PRRSV-2
Monocytes were transfected with 2 µM TGFbAS1 for 4 h.The TGFbAS1 mixture was removed, and monocytes were washed.TGFbAS1-transfected monocytes were inoculated with 100 µl of either cPRRSV-2 or HP-PRRSV-2 equivalent to a multiplicity of infection (m.o.i.) of 1.At the time of PRRSV-2 inoculation, each monocyte culture simultaneously received 100 µl of PBL (1 x 10 6 cells) from the same animal.The cultures were incubated for 48 h (37°C, humidified 5% CO 2 ), then received 50 µl of inducers.The cultures were incubated further for 12 h (37°C, humidified 5% CO 2 ).Cells were then harvested prior to RNA isolation.Cell culture supernatants were collected for the determination of TGFb1 protein levels by ELISA.Expressions of immune-related genes were determined every 12 h by real-time PCR.Controls included cells receiving mock Ag plus inducers (mock control); cells receiving PRRSV-2 and inducers (PRRSV-2-inoculated control); and cells treated with transfection media alone (without TGFbAS1), inoculated with PRRSV-2, and stimulated with inducers (PRRSV- Monocyte transfection and uptake (A) Monocytes under bright field microscopy.(B) Monocyte uptake of fluorescent-labeled siRNA under immunofluorescent microscopy.(C) Monocyte uptake of fluorescent-labeled siRNA complexed with 1.5% of transfection reagent.The uptake was observed every 2 h for 10 h and finally at 24 h.Mean differences of percentages of fluoresced cells 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.
inoculated/transfection media control).Untreated cells receiving culture media in the presence or absence of inducers served as positive and negative controls, respectively.Cell viability was determined at the end of the transfection period, PRRSV-2 inoculation, and inducer stimulation using trypan blue.
PRRSV-2 RNA was isolated and contaminating DNA was eliminated using PureLink ™ RNA mini kit (Invitrogen, USA).The quality and quantity of RNA were determined by OneDrop TOUCH Pro/Lite Micro-Volume Spectrophotometer (Biometrics Technologies Inc. USA).Reverse transcription (using the ReverTra Ace ® qPCR RT Kit, Toyobo, Japan) and real-time PCR were conducted as described previously (48).In brief, a total reaction volume of 20 µl, consisting of 2 µl cDNA, 10 µl SYBR ® Green PCR master mix (Toyobo), and 400 nM each of primers ORF7 149F and ORF7 346R was set up in duplicate.The PCR condition was 95°C (15 min); and 35 cycles of 95°C (15 s), 53°C (30 s), and 72°C (30 s).The C T was collected and compared with the standard curve of C T generated from 10 1 -10 8 copy numbers of recombinant PRRSV-2 ORF7 plasmids.Melting curve analysis and agarose gel electrophoresis were performed to verify a single product.Nuclease-free water was included as no template control in every run.
2.9 Evaluation of TGFb1 and IFNa protein effects on PRRSV RNA yields in monocytes and PBL co-culture inoculated with cPRRSV-2 and HP-PRRSV-2

Statistical analysis
Statistical analyses were performed using the SPSS software version 28 (IBM, Armonk, NY).Mean differences of immune-related gene expressions among groups were tested by one-way ANOVA, followed by Tukey HSD test.Mean differences of PRRSV-2 ORF7 copy numbers and immune-related gene expressions among groups at time points were tested by one-way repeated measures ANOVA, followed by Tukey HSD test.P<0.05 was set as a statistically significant level.

Efficient knockdown of TGFb1 mRNA expression by TGFbAS1
Tr. media of 1.5% (v/v) complexed with fluorescent-labeled siRNA control and a transfection period of 4 h had yielded the highest transfection efficiency with approximately 74% fluorescentpositive monocytes (Figure 1C).These conditions were therefore used for subsequent transfection experiments.Tr. media of 0.75% and 3% were also tested (Additional File 2) to confirm the effect of transfection media concentrations, in which 0.75% transfection media yielded the lowest transfection efficiency and 3% Tr. media had no signifi cant difference to 1.5% Tr. media in transfection efficiency.
TGFb1 mRNA expression (Figure 2A) and protein translation (Figure 2B) were efficiently downregulated by TGFbAS1 in ConAstimulated monocytes and PBL co-culture.The same downregulation by TGFbAS1 was observed in PMAi-stimulated monocytes and PBL co-culture (Figures 2C, D).TGFbAS1 targeted the AUG region of porcine TGFb1 mRNA and had no activitydecreasing motifs to ensure the antisense activity.Scr and Tr.media had no significant effect on TGFb1 mRNA expression when compared to positive control.

Specificity of TGFbAS1 knockdown
Using BLAST, the specificity of TGFbAS1 and Scr was evaluated and analyzed.TGFbAS1 was specific to porcine TGFb1 mRNA and had no aligned target in any porcine immune-related genes reported in this study or essential genes involved in the porcine immune system.Also, Scr had no aligned target in any of the porcine genes of interest.TGFbAS1 also had no aligned target in any ORFs of PRRSV-2 strains used in this study.
Slightly reduced mRNA expressions of FoxP3 (1.3 ± 0.3 vs 2.0 ± 0.4) and IL-10 (4.0 ± 0.2 vs 4.6 ± 0.3) as compared to positive control were observed in TGFbAS1-transfected monocytes co-cultured with PBL (Table 1).On the other hand, slightly increased mRNA expressions of IFNg (5.5 ± 0.2 vs 4.8 ± 0.2), MHC-I (3.1 ± 0.3 vs 2.4 ± 0.4), and STAT2 (2.4 ± 0.3 vs 1.9 ± 0.4) were demonstrated in TGFbAS1-transfected monocytes then co-cultured with PBL, when compared to positive control.But these changes in mRNA expression levels were not statistically significant.No significant changes in immune-related mRNA expression levels were observed in Scr and Tr.Media controls as compared to positive control.
No significant difference was observed with the mRNA expressions of FoxP3, GATA3, IL-2, IL-6, IL-17, and STAT6 between TGFbAS1+cPRRSV-2/HP-PRRSV-2-treated and cPRRSV-2/HP-PRRSV-2-inoculated monocytes and PBL co-culture.Transfection media has no significant effect on the expression levels of these immune-related genes as compared to positive control.
Compared to HP-PRRSV-2-inoculated monocytes cocultured with PBL, rTGFb1-treated (10 ng/ml final) monocytes inoculated with HP-PRRSV-2 and then co-cultured with PBL displayed significantly higher amount of PRRSV-2 ORF7 RNA copy numbers at 12, 24, 36, 48, and 60 h after inoculation (Figures 6A, B).Monocytes treated with rTGFb1 (1 and 0.1 ng/ ml final) and then inoculated with HP-PRRSV-2 prior to the coculture of PBL did not demonstrate a lower amount of PRRSV-2 ORF7 RNA copy numbers after inoculation.
Compared to cPRRSV-2-inoculated monocytes co-cultured with PBL, monocytes treated with rTGFb1, followed by rIFNa prior to cPRRSV-2 inoculation and PBL co-culture showed no alteration of the amount of PRRSV-2 ORF7 RNA copy numbers after inoculation (Figures 8A, B).Likewise, compared to HP-PRRSV-2-inoculated monocytes co-cultured with PBL, monocytes treated with rTGFb1, followed by rIFNa prior to HP-PRRSV-2 inoculation and PBL coculture did not show alteration of the amount of PRRSV-2 ORF7 RNA copy numbers after inoculation (Figures 8A, B).No PRRSV-2 ORF7 RNA was detected in cells treated with mock Ag.

Discussion
The effects of PRRSV-induced TGFb1 overexpression on mRNA expressions of transcription factors, type I and II IFNs, MHCs, anti-inflammatory and pro-inflammatory cytokines in PRRSV-2-inoculated cells were investigated in this study.PRRSV-2-induced TGFb1 overexpression in cells e.g., monocytes, MDMs, and in lungs and lymphoid organs of pigs has been reported (27,34,35).At present, the detailed function of PRRSV-2-up-regulated TGFb1 expression on swine immune protection against PRRSV-2 has not yet been explored.
The phosophorothioate-modified TGFbAS ODN targeting the AUG region of TGFb1 mRNA significantly reduced TGFb1 mRNA expression and protein translation (Figure 2, Additional File 3).In swine immune setting, the AUG region is reported as the most efficient target for gene knockdown of at least cytokines i.e., IFNg, TGFb1, and IL-10 (49, 50).Theoretically, phosophorothioatemodified AS ODNs hybridize specifically to target region of mRNA, which enables RNaseH to cleave the hybridized target mRNA.This consequentially results in the degradation of the intact mRNA template for protein translation.
Porcine monocytes are permissive to PRRSV-2 infection in the PBMC population.In the previous research, adherent porcine monocytes in the well-plate had greater than 90% of SWC3a + , which is indicative of monocytes (44).Non-adherent cells in PBMCs consisting of PBLs incubated for 2 h has been demonstrated to yield CD3 + lymphocytes and with less than 5% of SWC3 + cells in the harvested population (26).To indicate Thelper lymphocyte polarization, transcription factors i.e., FOXP3, T-bet, GATA3, and RORgT, together with immune-related genes i.e., TGFb1, IFNg, IL4, and IL17, were chosen as representative indicators of Treg, Th1, Th2, and Th17, respectively.
TGFbAS1 transfection in monocytes then co-cultured with PBL and inoculated with cPRRSV-2 and HP-PRRSV-2 significantly reduced TGFb1 mRNA expression (Figures 3A, C).Percentage reductions of 41% and 38% were observed on TGFbAS1transfected/cPRRSV-2-inoculated cells then stimulated with ConA and PMAi, respectively.Also, there were 25% and 27% reductions in TGFbAS1-transfected/HP-PRRSV-2-inoculated cells then Effects of rTGFb1 on PRRSV-2 ORF7 RNA copy numbers in PRRSV-2-inoculated monocytes co-cultured with PBL.Monocytes were treated with rTGFb1 (10, 1 and 0.1 ng/ml final), inoculated with either cPRRSV-2 or HP-PRRSV-2, then co-cultured with PBL (0 h), and finally stimulated with inducers i.e., (A) ConA or (B) PMAi (48 h).Monocytes inoculated with cPRRSV-2 or HP-PRRSV-2, then co-cultured with PBL and stimulated with inducers served as PRRSV-2-inoculated control.Monocytes receiving mock Ag then co-cultured with PBL plus inducer 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 10 1 -10 8 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.
stimulated with ConA and PMAi, respectively.ConA and PMAi were used to induce mRNA expressions of TGFb1 and other immune-related genes of interest.In the presence of these inducers, we clearly demonstrate that PRRSV-2 suppresses mRNA expressions of several immune-related genes in PRRSVinoculated cells as compared to uninoculated control (45, 49).Unexpectedly, mRNA expressions of IL-4 and IL-10 were significantly reduced with transfection of TGFbAS1 in monocytes co-cultured with PBL and inoculated with cPRRSV-2 and HP-PRRSV-2 (Figure 4, Additional File 4).The significant reductions of IL-4 and IL-10 mRNA expressions were not detected in the specificity testing of TGFbAS1 in uninoculated monocytes and PBL co-culture (Table 1).Approximately, there were 18% and 11% reductions of IL-4 and IL-10 in TGFbAS1-transfected/ cPRRSV-2-inoculated cells, respectively.Whereas, TGFbAS1transfected/HP-PRRSV-2-inoculated cells had approximately 22% and 5% reductions in IL-4 and IL-10 mRNA expressions, respectively.The outcomes of reduced IL-4 and IL-10 expressions in TGFbAS1-transfected/PRRSV-2-inoculated monocytes and PBL co-culture were not clearly understood.IL-10 and TGFb reportedly promote each other's gene expression (70).In human monocytes and macrophages, TGFb induces IL-10 production (71).In murine T-cells, TGFb suppresses Th2 by inducing T-regs (72).IL-4 primarily drives Th2 polarization.Th1 cells produce IFNg that inhibits IL-4-mediated Th2 differentiation (73).In pigs, IL-4 expression has been shown to control APC's inflammatory activities.IL-4 was recorded to induce porcine PAMs to become an alternatively activated M2 macrophages that are characterized by IL-10 production (74).In both human and murine, IL-4 is vital for antibody production and a diagnostic marker for Th2 immune cell response.In contrast, IL-4 is not a porcine B-cell's stimulatory factor because it impedes IL-6 and antibody secretion, and Effects of rIFNa on PRRSV-2 ORF7 RNA copy numbers in PRRSV-2-inoculated monocytes co-cultured with PBL.Monocytes were treated with rIFNa (10, 1 and 0.1 ng/ml final), inoculated with either cPRRSV-2 or HP-PRRSV-2, then co-cultured with PBL (0 h), and finally stimulated with inducers i.e., (A) ConA or (B) PMAi (48 h).Monocytes inoculated with cPRRSV-2 or HP-PRRSV-2, co-cultured with PBL, and finally stimulated with inducers served as PRRSV-2-inoculated control.Monocytes receiving mock Ag then co-cultured with PBL plus inducer 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 10 1 -10 8 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.
suppresses antigen-stimulated proliferation of B-cells (75).IL-4 was also demonstrated to suppress the transcription of inflammatory cytokines in porcine macrophages (76).The slightly reduced IL-4 and IL-10 after TGFb knockdown might be attributed to the upregulation of STAT1/2 and IFNa/g.This hypothesis needs to be verified by more studies, as no significant differences in T-bet expression were observed in PRRSV-2-inoculated and TGFbAS1transfected/PRRSV-2-inoculated monocytes and PBL co-culture.
The transfection of monocytes with TGFbAS1, followed by coculturing with PBLs and subsequent inoculation with cPRRSV-2 and HP-PRRSV-2, resulted in a significant decrease in PRRSV-2 ORF7 RNA copy numbers (Figure 5).This reduction was observed from 12 h to 60 h after inoculation.In TGFbAS1-transfected/ cPRRSV-2-inoculated cells, the reduction percentages of PRRSV-2 ORF7 RNA copy numbers were approximately 10.8% at 12 h after inoculation and 28.5% at 60 h after inoculation.Similarly, in TGFbAS1-transfected/HP-PRRSV-2-inoculated cells, the reduction percentage was approximately 9.6% at 12 h after inoculation and 24.6% at 60 h after inoculation.It is important to note that the reduction in PRRSV-2 ORF7 RNA copy numbers was not attributed to non-specific binding of TGFbAS1 to PRRSV RNA, as there was no alignment between TGFbAS1 and any ORFs of cPRRSV-2 and HP-PRRSV-2 used in this study.Furthermore, it is worth mentioning that the reduction percentage of PRRSV-2 ORF7 RNA copy numbers and TGFb1 mRNA expression was higher in TGFbAS1-transfected/cPRRSV-2-inoculated monocytes and PBL co-culture compared to TGFbAS1-transfected/HP-PRRSV-2inoculated monocytes and PBL co-culture.
In addition to the notable decrease in TGFb1 mRNA expression, a significant decrease in PRRSV-2 ORF7 RNA copy numbers was observed in TGFbAS1-transfected/cPRRSV-2-inoculated and TGFbAS1-transfected/HP-PRRSV-2-inoculated monocytes and PBL co-culture, accompanied by a significant increase in mRNA expressions of IFNa, IFNg, MHC-I, MHC-II, STAT1, and STAT2.Previous studies have reported the inhibitory effects of certain immune-related genes, such as IFNa, IFNg, and TNFa, on PRRSV replication (80)(81)(82).To further investigate the role of these immunerelated genes in reducing PRRSV-2 ORF7 RNA copy numbers, commercially available rIFNa was utilized.Treatment of monocytes and PBL co-culture with an optimal concentration of rIFNa prior to either cPRRSV-2 or HP-PRRSV-2 inoculation resulted in a significant reduction in PRRSV-2 ORF7 RNA copy numbers (Figure 7).Monocytes were susceptible to PRRSV-2 infection in the co-culture system.Knockdown of TGFb1 in monocytes within the co-culture system led to the upregulation of IFNa, IFNg, MHC-I, MHC-II, STAT1, and STAT2, which could potentially act as the anti-PRRSV response.These findings suggest that the significant increase in the expressions of these immunerelated genes in response to TGFb1 knockdown may contribute to the reduction of PRRSV-2 ORF7 RNA copy numbers.
rTGFb1 was employed to provide additional clarification regarding the potential role of PRRSV-up-regulated TGFb1 expression in supporting PRRSV replication.The treatment of monocytes and PBL co-culture with rTGFb1 prior to inoculation with either cPRRSV-2 or HP-PRRSV-2 resulted in a significant increase in PRRSV-2 ORF7 RNA copy numbers (Figure 6).Additionally, the treatment of cells with rTGFb1 prior to rIFNa treatment and inoculation with cPRRSV-2 or HP-PRRSV-2 led to a reduction in the antiviral activity of rIFNa (Figure 8).These findings demonstrate the positive influence of TGFb1 on PRRSV replication.Furthermore, these findings suggest a potential strategy employed by PRRSV to enhance virus replication and diminish innate immune defense against the virus through the upregulation of TGFb1 expression.

Conclusion
In conclusion, both cPRRSV-2 and HP-PRRSV-2 significantly upregulated the expression of TGFb1 in the co-culture of monocytes and PBL.The knockdown of TGFb1 expression by TGFbAS1 significantly enhanced the IFNa/g, MHC-I/II, and STAT1/2 mRNA expression levels in the monocytes and PBL cocultures infected with the virus.Additionally, the suppression of TGFb1 expression by TGFbAS1 contributed to the significant reduction in the yields of PRRSV-2 RNA copy numbers.Conversely, rTGFb1 and rIFNa sustained and decreased the yields of PRRSV-2 RNA copy numbers, respectively.The results of this study illustrate a plausible strategy employed by PRRSV to suppress the innate immune response, highlighting the immunomodulatory role of PRRSV-induced TGFb in dampening the innate immune defense against the virus.Furthermore, these findings suggest that the development of future PRRSV vaccines and vaccine adjuvants should consider targeting TGFb as a potential therapeutic approach.

2
FIGURE 2 Effect of TGFb1 antisense (TGFbAS1), and scramble (Scr) phosphorothioate-modified ODNs on expression of TGFb1 mRNA and protein translation in monocyte and PBL co-culture.Monocytes were transfected with TGFbAS1 or Scr, added with PBL, and then stimulated with either (A, B) ConA or (C, D) PMAi.Monocytes transfected with transfection media (Tr.media) alone, added with PBL, and finally stimulated with inducers (either ConA or PMAi) served as Tr.media control.Untransfected monocytes, added with PBL, and finally stimulated with inducers served as positive control (Pos Ctrl).Cell supernatant was collected prior to ELISA.Data were normalized to the geometric average of RPL32 and YWHAZ in relative to untransfected/unstimulated monocytes and PBL co-culture.Band intensities indicate the quality of TGFb1 knockdown (Additional File 3).Error bars indicate the standard deviation (SD).Mean differences of TGFb1 gene expression or protein translation among groups were tested by one-way ANOVA, followed by Tukey HSD test.Different letters above the error bars indicate significant differences.Data are presented in log 2 scale of "fold" according to 2^(-DDC T ) method.

3
FIGURE 3Effect of TGFbAS1 on TGFb1 mRNA expression and protein translation in PRRSV-2-inoculated PBMCs.Monocytes were transfected with TGFbAS1, then co-cultured with PBL, inoculated with either cPRRSV-2 or HP-PRRSV-2, and stimulated with inducers of either (A, B) ConA or (C, D) PMAi.Untransfected monocytes, co-cultured with PBL and inoculated with cPRRSV-2 or HP-PRRSV-2, stimulated with inducers, served as the PRRSV-2inoculated control.Monocytes treated with transfection media (Tr.media), co-cultured with PBL, and inoculated with cPRRSV-2 or HP-PRRSV-2, then stimulated with inducers served as PRRSV-2-inoculated/Tr.media control.Untransfected monocytes, co-cultured with PBL, inoculated with mock Ag, and stimulated with inducers served as mock control.Untreated monocytes then co-cultured with PBL and receiving culture media in the presence or absence of inducers served as positive and negative controls, respectively.(B, D) Cell culture supernatants were collected for ELISA.Error bars indicate the SD.Mean differences of TGFb1 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.
induce IL-10 production in vivo and ex vivo.The increased expression of TGFb1 in PRRSV-infected MDMs in vitro, and in lungs, lymph nodes and tonsils of PRRSV-infected pigs has been reported

TABLE 1
Expression levels of immune-related genes in monocytes transfected with either TGFbAS1 or Scr1, or otherwise treated with transfection media (Tr.media) alone prior to simultaneous addition of PBL for co-culture and stimulated with inducers, either ConA or PMAi.