Porcine Circovirus Type 2 Suppresses IL-12P40 Induction Via Capsid/Gc1qr-Mediated Micrornas and Signalings This Information Is Current As of September 27, 2021

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Porcine Circovirus Type 2 Suppresses IL-12p40 Induction via Capsid/gC1qR-Mediated MicroRNAs and Signalings This information is current as of September 27, 2021. Qian Du, Xingchen Wu, Tongtong Wang, Xuefeng Yang, Zhenyu Wang, Yingying Niu, Xiaomin Zhao, Shan-Lu Liu, Dewen Tong and Yong Huang J Immunol published online 1 June 2018 http://www.jimmunol.org/content/early/2018/05/31/jimmun Downloaded from ol.1800250

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 1, 2018, doi:10.4049/jimmunol.1800250 The Journal of Immunology

Porcine Circovirus Type 2 Suppresses IL-12p40 Induction via Capsid/gC1qR-Mediated MicroRNAs and Signalings

Qian Du,*,1 Xingchen Wu,*,1 Tongtong Wang,*,1 Xuefeng Yang,* Zhenyu Wang,* Yingying Niu,* Xiaomin Zhao,* Shan-Lu Liu,†,‡,x,{ Dewen Tong,* and Yong Huang*

Porcine circovirus (PCV) type 2 (PCV2), an immunosuppression pathogen, is often found to increase the risk of other pathogenic infections. Yet the relative immune mechanisms determining the susceptibility of PCV2-infected animals remain unclear. In this study, we conﬁrmed that PCV2 infection suppressed IL-12p40 expression and host Th1 immune response, leading to a weakened pathogenic clearance upon porcine reproductive respiratory syndrome virus (PRRSV) or Haemophilus parasuis infection. PCV2 infection suppressed pathogens, LPS/IFN-g, or LPS/R848-induced IL-12p40 expression in porcine alveolar macrophages. PCV2 capsid (Cap) was the major component to suppress IL-12p40 induction by LPS/IFN-g, LPS/R848, PRRSV, or H. parasuis. Either wild-type PCV2 or mutants

PCV2–replicase 1 and PCV type 1–Cap2, which contained PCV2 Cap, signiﬁcantly decreased IL-12p40 levels and increased the Downloaded from replication of PRRSV and H. parasuis in the lung tissues relative to mock or PCV type 1 infection. gC1qR, a Cap binding protein, was not involved in IL-12p40 induction but mediated the inhibitory effect of PCV2 Cap on IL-12p40 induction. PCV2 also activated PI3K/Akt1 and p38 MAPK signalings to inhibit IL-12p40 expression via inhibition of NF-kB p65 binding to il12B promoter and upregulation of miR-23a and miR-29b. Knockdown of Akt1 and p38 MAPK downregulated miR-23a and miR-29b and increased IL- 12p40 expression. Inhibition of miR-23a and miR-29b attenuated the inhibitory effect of PCV2 on IL-12p40 induction, resulting in an

increased IL-12p40 expression and Th1 cell population and reduced susceptibility to PRRSV or H. parasuis. Taken together, these results http://www.jimmunol.org/ demonstrate that PCV2 infection suppresses IL-12p40 expression to lower host Th1 immunity to increase the risk of other pathogenic infection via gC1qR-mediated PI3K/Akt1 and p38 MAPK signaling activation. The Journal of Immunology, 2018, 201: 000–000.

orcine circovirus (PCV) is a nonenveloped ssDNA virus, pathogen of PCV-associated disease (PCVAD), which is among which contains the nonpathogenic PCV type 1 (PCV1) the most economically significant diseases wasting the global P and pathogenic PCV type 2 (PCV2) (1, 2). PCV has two swine industry today (2). PCVAD is a multifactorial disease that is major open reading frames (ORF), ORF1 encoding the replicase usually observed in a coinfection of PCV2 with other patho- of virus (Rep) and ORF2 encoding the only capsid (Cap) (1). Rep gens, such as porcine reproductive respiratory syndrome virus by guest on September 27, 2021 is the most conserved protein between PCV1 and PCV2, whereas (PRRSV), porcine parvovirus, or Haemophilus parasuis (3, 4). Cap is significantly divergent (1). PCV2 is the primary causative PCV2 infection is required for the occurrence of PCVAD, but PCV2 infection alone rarely produces the full spectrum or severity of clinical disease (5, 6). Thus, PCV2 infection is considered to *College of Veterinary Medicine, Northwest A&F University, Yangling, China 712100; †Center for Retrovirus Research, The Ohio State University, Columbus, affect the host immune system, which leads to increased suscep- OH 43210; ‡Viruses and Emerging Pathogens Program, Infectious Diseases Institute, tibility in PCV2-infected animals (7). The Ohio State University, Columbus, OH 43210; xDepartment of Veterinary Bio- { In response to the attack of microbial pathogens, IL-12, as a key sciences, The Ohio State University, Columbus, OH 43210; and Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210 proinflammatory cytokine, plays a pivotal role in the generation of 1Q.D., X.W., and T.W. contributed equally to this work. Th1 immune response for combating pathogen infection (8, 9). IL-12 Received for publication February 22, 2018. Accepted for publication May 3, 2018. is a 70-kDa heterodimeric cytokine composed of p35 and p40 sub- This work was supported by the National Natural Science Foundation of units and produced by APCs, including monocytes/macrophages, China (Grant 31672535) and the U.S. National Institutes of Health (Grants dendritic cells, and B cells (10). IL-12p40 regulation is considered 1R01AI112381 and 1R21AI109464) to S.-L.L. This work was also supported by to be more critical for IL-12 production, comparing to IL-12p35 that the Science and Technology Innovation Project in Shaanxi Province (Grant 2016KTCL02-13), the Central Project of Major Agricultural Technology Promotion can’t be secreted without binding to IL-12p40 (11, 12). Thus, Funds (Grant K3360217060), and the Fundamental Research Funds for the Central IL-12p40 seems to play a more dominant role in promoting Th1 cell Universities (Grant 2452017023). development (13, 14). Several studies have proved that PCV2 in- Address correspondence and reprint requests to Prof. Dewen Tong and Prof. Yong Huang, Northwest A&F University, No. 22 Xinong Road, Yangling, China 712100. fection inhibits the IL-12p40 expression both in vivo and in vitro E-mail addresses: [email protected] (D.T.) and [email protected] (15–18). However, the molecular mechanisms underlying PCV2 (Y.H.) inhibition of IL-12p40 expression remain to be determined. The online version of this article contains supplemental material. In this study, we first determined the Th1 immune response and Abbreviations used in this article: Cap, capsid; miRNA, microRNA; MOI, multiplic- IL-12p40 production of PCV2-infected piglets challenged with ity of infection; ORF, open reading frame; PAM, porcine alveolar macrophage; PAMP, pathogen-associated molecular pattern; PCV, porcine circovirus; PCV1, PRRSV or H. parasuis and confirmed that PCV2 infection sup- PCV type 1; PCV2, PCV type 2; PCVAD, PCV-associated disease; PRRSV, porcine pressed the host Th1 immune response to PRRSV or H. parasuis reproductive respiratory syndrome virus; qPCR, quantitative PCR; rAd-Blank, through the inhibition of IL-12p40 induction in macrophages. recombinant adenovirus expressing blank control; rAd-Cap, recombinant adenovirus expressing PCV2 Cap; rAd-Rep, recombinant adenovirus expressing PCV2 Rep; Then we further analyzed and determined the correlation of Rep, replicase of virus; si-Akt1, Akt1 siRNA; siRNA, small interfering RNA; IL-12p40 induction, Th1 cell percentage, and pathogenic clear- TCID , 50% tissue culture infective dose; UTR, untranslated region. 50 ance and identified the roles of PCV2 Rep and Cap in its sup- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 pression of IL-12p40 expression. Results showed that PCV2 Cap

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800250 2 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs plays a predominant role in inhibition of IL-12p40 expression For small interfering RNA (siRNA) transfection, PAMs seeded in six- induced by other pathogens or TLR agonists in PCV2-infected well plates were transfected with 100 nM Akt1, p38, or ERK1-specific porcine alveolar macrophages (PAMs). PCV2 Cap binding pro- siRNAs for 24 h. Then the cells were infected by PCV2 and followed by LPS/IFN-g stimulation. The siRNA used in this work were targeted tein gC1qR, PCV2-activated PI3K/Akt1, and p38 MAPK signaling, to Akt1 (NM_001159776.1), p38 (XM_001929490.5), and ERK1 as well as upregulated miR-23a and miR-29b, are the key regulators (NM_001198922.1), respectively. The Akt1 siRNA (si-Akt1) sequence in PCV2 inhibition of IL-12p40. These results would be helpful to was 59-AACGAGGCGAGTACATCAAGA-39. The p38 siRNA (si-p38) explain how PCV2 infection suppresses IL-12 production to in- sequence was 59-AAGCTATCCAGACCATTTCAA-39. And the ERK1 siRNA (s-ERK1) sequence was 59-AAGCTCTTGAAGACGCAGCAC-39. crease the risk of other infections. ELISA Materials and Methods The supernatants of treated cells were harvested, and the levels of IL-12p40 and Ethics statement IFN-g secretion were measured by commercial ELISA kit (P1240 [R&D Systems] and 430101[BioLegend]) according to the manufacturers’ instructions. All animal experiments were approved by the Institutional Animal Care and Use Committee of Northwest A&F University (permit numbers: 20161013 Flow cytometry and 20170924) and were performed according to the Animal Ethics Pro- cedures and Guidelines of the People’s Republic of China. No other The PBMCs and PAMs were harvested and washed by PBS, and then the specific permissions were required for these activities. This study did not surface and intracellular proteins of the cells were stained according to the involve endangered or protected species. manufacturer’s illustration. Briefly, the washed cells were resuspended with staining buffer, and conjugated Abs were added to stain the surface Cells, viruses, and reagents proteins. Then the cells were fixed and permeabilized to stain the intracellular proteins with the conjugated Abs. After the wash process, the Downloaded from PK-15 cells, Marc-145 cells, and 3D4/21 cells (CRL-2843) were purchased stained cells were analyzed using BD Accuri C6 Flow Cytometer, and the from American Type Culture Collection and maintained in our laboratory. gating strategies were shown in Supplemental Fig. 1. The primary PAMs were obtained from the lungs of piglets as previously described (19). PBMCs were prepared by centrifugation over Lymphocyte qPCR Separation Medium (P8610; Solarbio) per the manufacturer’s instructions. PK-15 cells and Marc-145 cells were maintained in DMEM (12100046; The qPCR was performed as previously described (19). Briefly, the total RNA Invitrogen) supplemented with 10% heat-inactivated FBS (13011-8611; of the cells was isolated by TRIzol (Invitrogen); the reverse transcriptions Tianhang Biotechnology). 3D4/21 and primary PAMs were cultured in were performed with random primers or miRNA-specific primers. The ex- http://www.jimmunol.org/ RPMI 1640 medium (31800022; Invitrogen) with 10% heat-inactivated pression levels were measured by SYBR green–based real-time PCR using a FBS, sodium pyruvate (11360070; Life Technologies), nonessential Bio-Rad IQ5 Real-Time PCR System. The primer sequences for PCV1 were 9 9 9 amino acids (11140050; Life Technologies), 100 U/ml penicillin, and 5 -TCTTTCGGCGCCATCTGTAA-3 and 5 -CAGCAGCGCACTTCTTT- 9 9 0.1 mg/ml streptomycin. PCV1 (AY193712) and PCV2 (EU366323) were CAC-3 . The primers for PCV2 were 5 -ATAACCCAGCCCTTCTCCTACC- 9 9 9 stocked in our laboratory and propagated in PK-15 cells. PRRSV 3 and 5 -GGCCTACGTGGTCTACATTTCC-3 . The primers for IL-12p40 9 9 9 (FJ548855.1) previously isolated by our team was propagated in Marc-145 (NM_214013.1) were 5 -TGTTCAAGTTCAGGGCAAGA-3 and 5 -CAG- 9 cells (3). The copy numbers of the viruses were quantified by real-time GAGGAGCTGTAGTAGCG-3 . The miRNA primers were presented in Supplemental Table 1. PCR, and the 50% tissue culture infective dose (TCID50) was measured by the Reed–Muench method. H. parasuis was isolated from the native herd Luciferase reporter assays

and cultured with Tryptone soya agar (22091; Sigma-Aldrich) and Tryp- by guest on September 27, 2021 tone soya broth (51228; Sigma-Aldrich), supplemented with 2% b-NAD The PAMs were transfected with the reporter vector containing NF-kB trihydrate (NAD, N7004; Sigma-Aldrich) and 5% FBS. response elements based on pGL4 (pGL4.32[luc2P/NF-kB-RE/Hygro]) The TLR agonists LPS (L2880) and R848 (tlrl-r848) were purchased from and the normalizing control vector pRL Renilla luciferase (Promega), to- Sigma-Aldrich and InvivoGen, respectively. The IFN-g (985-PI) was pur- gether with specific siRNAs for Akt1, p38, ERK1, or negative control, and chased from R&D Systems. The flow cytometry buffers, monensin, and then infected by mock or PCV2 for another 24 h, followed by LPS/IFN-g APC-conjugated anti–IFN-g (502511, 4S.B3), FITC-conjugated anti-CD4 stimulation. At 24 h poststimulation, the luciferase activities were deter- (317408, OKT4), APC-conjugated anti-CD68 (333810, Y1/82A), and PE- mined with Dual-Luciferase reporter systems (Promega) according to the conjugated anti–IL-12p40 (501807, C11.5) were purchased from BioLegend. manufacturer’s instructions. Animal, housing, and experiment design Macrophage and PBMC cocultures 6 Six-week-old cross-bred piglets were purchased from a native herd free of The PAMs were seeded into six-well plates (1 3 10 cells per well) and PCV2, PRRSV, porcine parvovirus, H. parasuis, and other major swine transfected with miRNA-specific inhibitors, and then the cells were in- pathogens as determined by PCR and ELISA. All piglets were housed fected with PCV2 or mock for 24 h. Then PAMs were incubated with under the same conditions and treated in a similar way. For the first ex- PBMC isolated from healthy piglets in a 1:1 ratio and further challenged periment, 30 piglets were randomly divided into three groups and inocu- with PRRSV or H. parasuis for 24 h. PBMC and the PAMs were harvested lated with PCV1 (4 3 105 TCID ), PCV2 (4 3 105 TCID ), or mock from the coculture by gentle pipetting and resuspended in 1 3 PBS. The 50 50 + (same volume DMEM) for 1 wk, respectively. Then the pigs were further IL-12p40 expression by PAMs and IFN-g expression by CD4 T cells were 5 8 challenged with PRRSV (10 TCID50)orH. parasuis (10 CFU) for an- analyzed by flow cytometry, and the replication of PRRSV and H. parasuis other 24 h. For the second experiment, another 70 piglets were divided into were measured. 5 5 seven groups to inoculate PCV1 (4 3 10 TCID50), PCV2 (4 3 10 5 5 Statistical analysis TCID50), PCV1 mutants (4 3 10 TCID50), PCV2 mutants (4 3 10 TCID50), or mock (same volume DMEM), respectively, and further chal- 5 8 The results are representative of three independent experiments. The data are lenged with PRRSV (10 TCID50)orH. parasuis (10 CFU). The PAMs presented as mean 6 SEM (SD). Comparisons between the two groups were and PBMCs of the piglets were prepared for further analysis. performed by unpaired Student t test, whereas multiple group data were analyzed by ANOVA, followed by Bonferroni post hoc test. Statistically sig- Infection and stimulation of PAMs nificant and very significant results were defined as p , 0.05 and p , 0.01. PAMs were seeded into six-well plates at 1 3 106 cells per well and were mock-infection, PCV1 mutants infection (multiplicity of infection [MOI] Results of 1), PCV2 mutants infection (MOI of 1), PCV1 infection (MOI of 1), or PCV2 infection suppresses IL-12p40 expression and Th1 PCV2 infection (MOI of 1). At 24 h postinfection, the media were refreshed, and then the cells were challenged with PRRSV (MOI of 1) or immune response to promote PRRSV and H. parasuis infection H. parasuis (MOI of 1) or stimulated with LPS (1000 ng/ml) plus IFN-g in the lung of piglets (100 ng/ml) or LPS (1000 ng/ml) plus R848 (5 mg/ml). The supernatants of the cells were measured by ELISA, and the protein and mRNA levels of Th1 immune response derived by IL-12 plays a critical role in IL-12p40 and microRNA (miRNA) levels of the cells were measured by intracellular pathogen clearance (20, 21), and several studies have flow cytometry and quantitative PCR (qPCR), respectively. revealed that PCV2 infection affects IL-12 expression (15–18). To The Journal of Immunology 3

figure out how PCV2 infection influences IL-12 expression and produced in PBMCs isolated from mock- and PCV1-infected pig- the Th1 immune response of piglets, the piglets infected by PCV1 lets than from PCV2-infected piglets (Fig. 1B). Likewise, in lung or PCV2 were challenged by PRRSV or H. parasuis to test the and pulmonary lymph nodes, the levels of IL-12p40 and IFN-g and effects of PCV2 infection on the expression of IL-12p40 and the percentage of CD4+INF-g+ Th1 cells were higher in mock- and IFN-g and the activation of Th1 cell subsets. Upon PRRSV or PCV1-infected pigs than in PCV2-infected pigs after PRRSV or H. parasuis challenge, serum IL-12p40 and IFN-g were more H. parasuis challenge (Fig. 1C, 1D), These results suggest that significantly upregulated in mock- and PCV1-infected pigs than in PCV2 infection suppresses IL-12p40 expression and the host Th1 PCV2-infected pigs (Fig. 1A); more CD4+INF-g+ Th1 cells were immune response to the secondary infection in the piglets. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

5 FIGURE 1. PCV2 infection suppresses IL-12p40 induction and Th1 immune response of piglets. The piglets were infected by PCV1 (4 3 10 TCID50), 5 5 8 PCV2 (4 3 10 TCID50), or mock (same volume of medium) for 1 wk, respectively, and then challenged with 10 TCID50 PRRSV or 10 CFU H. parasuis for another 24 h. (A) The serum IL-12p40 and IFN-g of the infected piglets were measured by ELISA. (B) The PBMC of the infected piglets were isolated and strained with CD4 and IFN-g mAbs to measure the CD4+IFN-g+ Th1 cells. (C) The expression of IL-12p40 and IFN-g was measured in lung by ELISA. (D) The CD4+IFN-g+ Th1 cells in pulmonary lymph nodes were measured by ﬂow cytometry. (E) The IL-12p40 expression of CD68-positive PAMs was analyzed by ﬂow cytometry in the PAMs from mock-, PCV1-, or PCV2-infected piglets upon PRRSVand H. parasuis challenges. (F) The replication of PRRSV and H. parasuis in the lungs was measured. (G) The correlation of the replication of PRRSV and H. parasuis with the percentages of IL-12p40– or IFN-g–positive cells in different dosages of PCV2-infected piglets were analyzed. The results are mean 6 SEM of three independent experiments or mean 6 SD representative of three independent experiments. *p , 0.05, **p , 0.01 versus mock infection cells. #p , 0.05, ##p , 0.01 versus PCV1-infected cells in (A) and (C). 4 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs

Next, we tested and compared the IL-12p40 expression of PAMs 3A, 3B). Although the expression of IL-12p40 induced by LPS/ isolated from mock-, PCV1-, and PCV2-infected piglets. Results IFN-g or LPS/R848 was lower in rAd-Cap–infected cells compared showed that PRRSV and H. parasuis challenge induced more with rAd-Rep–infected cells (Fig. 3A, 3B), these results suggest IL-12p40–positive cell production (64.0, 56.6%) in the CD68+ that PCV2 Cap can more strongly inhibit IL-12p40 induction rel- macrophages from mock-infected pigs than that induced in the ative to Rep protein. CD68+ macrophages from PCV2-infected piglets (24.2, 15.5%), To further confirm the roles of Cap and Rep in the inhibition of whereas PCV1-infected piglets showed a similar change as mock- IL-12p40 expression, we constructed PCV1 mutants that replaced infected pigs in the percentages of CD68+IL-12p40+ PAMs upon ORF1 or ORF2 in PCV1 backbone by PCV2 ORF1 or ORF2 PRRSV and H. parasuis challenges (74.7, 58.8%) (Fig. 1E), sug- (named PCV1-Rep2 and PCV1-Cap2) and PCV2 mutants that gesting that PCV2 infection significantly suppresses the IL-12p40 replaced ORF1 or ORF2 in PCV2 backbone by PCV1 ORF1 or expression of PAMs upon secondary various infections but that ORF2 (named PCV2-Rep1 and PCV2-Cap1), respectively PCV1 does not. To further determine whether the PCV2 infection (Supplemental Fig. 2B). When PAMs were infected with PCV1, affects the replication of PRRSVor H. parasuis,thelungsofmock-, PCV2, PCV1 mutants, or PCV2 mutants, IL-12p40 was more PCV1-, or PCV2-infected piglets were collected, and the levels of markedly induced by PCV1, PCV1-Rep2, or PCV2-Cap1 infec- PRRSV or H. parasuis were detected. Results showed that the tion than PCV2, PCV1-Cap2, or PCV2-Rep1 infection TCID50 of PRRSV and CFU of H. parasuis were not significantly (Supplemental Fig. 2C). Upon LPS/IFN-g or LPS/R848 stimula- different between mock-infected and PCV1-infected pigs, whereas tion, IL-12p40 production was also apparently lower in PCV2-, PCV2 infection markedly promoted the replication of both PRRSV PCV1-Cap2–, and PCV2-Rep1–infected PAMs than in PCV1- or and H. parasuis relative to mock and PCV1 infection (Fig. 1F). mock-infected PAMs, whereas PCV1-Rep2 and PCV2-Cap1 in- Downloaded from Furthermore, Pearson correlation analysis showed the percentages fection did not show a significantly inhibitory effect on IL-12 of CD68+IL-12p40+ PAMs and CD4+INF-g+ Th1 cells were in- induction relative to PCV1 or mock infection (Fig. 3C, 3D). versely associated with the replication levels of PRRSV and Likewise, in the in vivo experiments, PRRSV or H. parasuis H. parasuis in piglets infected with different doses of PCV2 challenge induced a lower level of IL-12p40 in PCV2-, PCV1- (Fig. 1G). Together, these results demonstrate that PCV2 infection Cap2–, or PCV2-Rep1–infected piglets, when compared with

suppresses IL-12p40 expression and Th1 immune response to PCV1- or mock-infected piglets (Fig. 3E, 3F). Consistent with the http://www.jimmunol.org/ promote PRRSV and H. parasuis infection in the lung of piglets. difference of IL-12p40 in different PCV-infected piglets, the TCID50 of PRRSV and CFU of H. parasuis in the lung tissues PCV2 infection inhibits pathogens- or pathogen-associated from PCV2-, PCV1-Cap2–, or PCV2-Rep1–infected piglets were molecular pattern–induced IL-12p40 expression in PAMs both significantly increased compared with PCV1- or mock-infected piglets ex vivo and in vitro (Fig. 3G, 3H). Of note, in both PRRSV- and H. parasuis–challenged To further confirm that PCV2 infection could suppress IL-12p40 piglets, PCV2 infection more markedly inhibited IL-12p40 induc- induction, the PAMs isolated from mock-, PCV1-, and PCV2- tion and promoted the infection of PRRSV and H. parasuis relative infected pigs were cultured ex vivo for 24 h, and the cells were toPCV1andallPCV1andPCV2mutants,whereasPCV1-Cap2 further infected by PRRSV or H. parasuis or stimulated by and PCV2-Rep1 mutants showed stronger inhibitory effects on by guest on September 27, 2021 pathogen-associated molecular pattern (PAMP) molecules, such as IL-12p40 induction and stronger enhanced effects on PRRSV and bacterial LPS, TLR7/8 agonist (R848), and macrophage-activating H. parasuis infection than PCV1-Rep2 and PCV2-Cap1 mutants cytokine (IFN-g) to mimic the effect of RNA viruses and bacteria (Fig. 3E–H). These results further confirm that Cap protein plays a infection. PRRSV and H. parasuis infection did significantly induce predominant role in inhibition of IL-12p40 induction and pro- IL-12p40 expression in mock-, PCV1-, and PCV2-infected PAMs at moting the infection of other pathogens relative to Rep protein both protein and mRNA levels, but the upregulation of IL-12p40 in vivo, even though overexpression of Rep shows a certain in- was remarkably lower in PCV2-infected PAMs (Fig. 2A, 2B). hibitory effect on IL-12p40 induction in vitro. Meanwhile, LPS/IFN-g or LPS/R848 stimulation also significantly induces IL-12p40 in mock-, PCV1-, and PCV2-infected PAMs at Host gC1qR protein is crucial for the suppression of IL-12p40 both protein and mRNA levels, and PCV2 infection also efficiently induction in PCV2-infected PAMs inhibited the upregulation of IL-12p40 (Fig. 2C, 2D). Consistent In a previous study, we found that PCV2 infection induces IL-10 with the ex vivo results, PCV2-infected fresh PAMs also showed a production in PAMs by Cap and gC1qR interaction (19). To lower IL-12p40 expression relative to mock- or PCV1-infected determine whether gC1qR participates in the suppression of fresh PAMs when these cells were further infected with PRRSV IL-12p40 production in PCV2-infected cells, we compared the or H. parasuis or stimulated with LPS/IFN-g or LPS/R848 induction of IL-12p40 in the wild-type (gC1qR+/+) and (Fig. 2E–H). These results further confirm that PCV2 infection gC1qR-deficient (gC1qR2/2) PAMs that were generated in our suppresses pathogen-induced IL-12p40 expression in PAMs. previous work. In mock infection cells, gC1qR deficiency did not significantly affect LPS/IFN-g or LPS/R848-induced IL-12p40 PCV2 Cap plays a predominant role in the suppression of production, but in PCV2-infected cells, IL-12p40 levels was IL-12p40 expression and promoting the infection of other markedly higher in gC1qR2/2 cells than in gC1qR+/+ cells upon pathogens in vivo LPS/IFN-g stimulation (Fig. 4A). In other words, even though To make clear which component of PCV2 plays the critical roles in IL-12p40 levels were lower in PCV2-infected gC1qR+/+ cells than the suppression of IL-12p40 expression, PAMs were infected with in mock-infected gC1qR+/+ cells, there was no significant differ- the recombinant adenoviruses expressing PCV2 Rep (rAd-Rep), ence between PCV2-infected gC1qR2/2 cells and mock-infected PCV2 Cap (rAd-Cap), or blank control (rAd-Blank) for 24 h and gC1qR2/2 cells (Fig. 4A). A gC1qR deficiency also showed then stimulated by LPS/IFN-g or LPS/R848 to detect IL-12p40 similar effects on the PCV2-induced IL-12p40 inhibition in expression. Rep and Cap protein were expressed in the PAMs in- mRNA levels in PAMs (Fig. 4B), suggesting that gC1qR is infected with rAd-Rep and rAd-Cap, respectively (Supplemental Fig. volved in the PCV2-induced IL-12p40 inhibition. To further de- 2A) and markedly suppressed IL-12p40 expression induced by termine whether gC1qR is involved in Cap-mediated but not in LPS/IFN-g or LPS/R848 at both protein and mRNA levels (Fig. Rep-mediated IL-12p40 suppression, gC1qR+/+ and gC1qR2/2 The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. PCV2 infection suppresses the expression of IL-12p40 at both transcriptional and posttranscriptional levels in PAMs. (A–D) The PAMs were isolated from mock-, PCV1-, or PCV2-infected piglets. Then the cells were seeded into six-well plates and cultured for 24 h, further infected by 1 MOI PRRSV or 50 MOI H. parasuis for 6 or 24 h or stimulated by LPS/IFN-g (1000, 100 ng/ml) or LPS/R848 (1000 ng/ml, 5 mg/ml) for 6 or 24 h. The protein and mRNA levels of IL-12p40 were measured by ELISA and qPCR, respectively. (E–H) The PAMs from healthy pigs (Figure legend continues) 6 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs

PAMs were infected with rAd-Rep or rAd-Cap. Following by and miR-29b expression in PAMs, and PCV2 Cap is the key LPS/IFN-g stimulation, IL-12p40 protein and mRNA did not compound for upregulation of these miRNA expressions. show significant difference between gC1qR+/+ and gC1qR2/2 gC1qR, Akt1, and p38 MAPK participate in the regulation PAMs after rAd-blank or rAd-Rep infection, but gC1qR2/2 PAMs of miR-23a, miR-23b, miR-29a, and miR-29b in were able to express more IL-12p40 than gC1qR+/+ PAMs after PCV2-infected PAMs rAd-Cap infection (Fig. 4C, 4D), suggesting that gC1qR is only involved in Cap-mediated IL-12p40 suppression in PCV2-infected Because PCV2 Cap could bind with gC1qR to activate PI3K/Akt1, PAMs. ERK, and p38 MAPK signaling and Cap was the key component of PCV2 to upregulate miR-23a, miR-23b, miR-29a, and miR-29b Akt1 and p38 MAPK are involved in the suppression of expression in PAMs, we checked out whether gC1qR, Akt1, IL-12p40 expression in PCV2-infected PAMs ERK1, and p38 MAPK participated in the regulation of miR-23a, PI3K/Akt1, ERK, and p38 MAPK pathways were activated by Cap miR-23b, miR-29a, and miR-29b in PCV2-infected PAMs. As the in PCV2-infected PAMs previously (19), and these pathways results show, PCV2-induced miR-23a, miR-23b, miR-29a, and might also be critical for the regulation of IL-12p40 expression in miR-29b upregulation were almost abolished in the gC1qR porcine macrophages (22). To determine the roles of these sig- knockout PAMs (Fig. 7A), suggesting gC1qR is involved in me- nalings in PCV2 inhibition of IL-12p40 expression, the specific diating the upregulation of four miRs in PCV2-infected cells. In siRNAs of Akt1, p38 MAPK, and ERK1 were used to down- the Akt1-specific siRNA-pretreated PAMs, PCV2-induced miR- regulate the protein expressions (Supplemental Fig. 2D); IL-12p40 23a, miR-23b, and miR-29b expression were inhibited, whereas expression was examined in these cells after PCV2 infection. miR-29a was not altered (Fig. 7B), suggesting Akt1 mainly me- Downloaded from Results showed that si-Akt1 treatment not only seemed to re- diates the upregulation of miR-23a, miR-23b, and miR-29b in markably improve the IL-12p40 expression in mock infection PCV2-infected cells. In the p38-specific siRNA-pretreated PAMs, PAMs in both protein and mRNA levels but also could markedly PCV2-induced miR-29a and miR-29b expression was reduced, decrease the inhibitory effects of PCV2 on IL-12p40 expression whereas miR-23a and miR-23b expression did not change upon LPS/IFN-g stimulation (Fig. 5A, 5B). Like the treatment of (Fig. 7C), suggesting p38 MAPK mainly mediates the upregula-

si-Akt1, the p38 MAPK–specific siRNA could also increase LPS/ tion of miR-23a and miR-23b in PCV2-infected cells. In the http://www.jimmunol.org/ IFN-g–induced IL-12p40 production in PCV2-infected PAMs in ERK1-specific siRNA-pretreated PAMs, all four miRNA levels both protein and mRNA levels, whereas the ERK1-specific siRNA were not largely altered when compared with nonspecific siRNA- did not affect the IL-12p40 expression in mock-infected cells or in pretreated PAMs (Fig. 7D), suggesting ERK signaling is not PCV2-infected cells (Fig. 5A, 5B). Furthermore, Akt1 and p38 involved in the regulation of these miRNAs. These results MAPK–specific siRNA treatment could efficiently increase the demonstrate that PCV2 binds with host gC1qR to regulate miR- binding of NF-kB p65 to the il12B (IL-12p40) promoter, whereas 23a and miR-23b expression through activation of PI3K/Akt1 ERK1-specific siRNA did not (Fig. 5C). In addition, the down- signaling, to regulate miR-29a expression via p38 MPAK signal- regulation of Akt1 and p38 MAPK significantly raised the NF-kB ing, and to regulate miR-29b expression via PI3K/Akt1 and p38 p65 activity in PCV2-infected cells (Fig. 5D). These results in- MAPK signaling. by guest on September 27, 2021 dicate that Akt1 and p38 MAPK participate in the suppression of PCV2 infection suppression of IL-12p40 induction mainly IL-12p40 expression in PCV2-infected PAMs, at least at the depends on miR-23a and miR-29b at posttranscriptional level transcriptional level. Because the miR-23a, miR-23b, miR-29a, and miR-29b were PCV2 Cap upregulates miR-23a, miR-23b, miR-29a, upregulated in PCV2-infected PAMs and might be related to the and miR-29b expression in PAMs IL-12p40 expression, we employed the mimics of these miRNAs to To further figure out whether PCV2 infection regulates IL-12p40 pretreat the PAMs and to detect the IL-12p40 expression upon LPS/ expression in posttranscriptional levels, the 12 miRNAs that were IFN-g or LPS/R848 stimulation. Results showed the miR-23a, predicted to regulate porcine IL-12p40 were analyzed in PCV2- miR-23b, miR-29a, and miR-29b mimics treatment significantly infected PAMs by qPCR. The results showed that the expression repressed the LPS/IFN-g or LPS/R848-induced IL-12p40 pro- levels of miR-23a, miR-23b, miR-29a, and miR-29b were sig- duction at protein level (Fig. 8A). Meanwhile, miR-29a and -29b nificantly upregulated in PCV2-infected PAMs compared with markedly reduce IL-12p40 mRNA levels when compared with the mock infection PAMs, especially miR-23a and miR-29b (Fig. 6A). negative mimic treatment but miR-23a and -23b did not (Fig. 8B). To find out which component of PCV2 regulated the miRNA These results suggest that miR-23a and miR-23b inhibit IL-12p40 expressions, the expression of these miRNAs was detected in the expression at the posttranscriptional level, whereas miR-29a and PCV2-, PCV2-Cap1–, or PCV2-Rep1–infected PAMs. Compared miR-29b likely inhibit IL-12p40 expression at both transcriptional with mock infection, PCV2 and PCV2-Rep1 infections signifi- and posttranscriptional levels. cantly upregulated miR-23a, miR-23b, miR-29a, and miR-29b To determine whether these miRNAs regulate IL-12p40 ex- expression in PAMs, yet PCV2-Cap1 infection did not signifi- pression at the posttranscriptional level, we constructed reporter cantly induce these miRNAs (Fig. 6B). Consistently, the rAd-Rep plasmids encoding the 39 untranslated region (UTR) of wild-type did not significantly upregulate miR-23a, miR-23b, miR-29a, and porcine il12B mRNA downstream of the firefly luciferase gene miR-29b expression in PAMs, whereas rAd-Cap could induce and parallel construct, including mismatches in the predicted these miRNA expressions (Fig. 6C). These results demonstrate binding sites (miR-23a, miR-23b, miR-29a, or miR-29b) of the that PCV2 infection can upregulate miR-23a, miR-23b, miR-29a, il12B 39UTR (Supplemental Fig. 3A). Reporter assays showed

were isolated and cultured for 24 h, and then the cells were infected by PCV1, PCV2, or mock for 24 h and further challenged by PRRSV or H. parasuis or stimulated by LPS/IFN-g or LPS/R848. The protein and mRNA levels of IL-12p40 expression were measured by ELISA and qPCR, respectively. *p , 0.05, **p , 0.01 versus mock-infected cells with same secondary infection or same stimulation. #p , 0.05, ##p , 0.01 versus PCV1-infected cells with same secondary infection or same stimulation. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. PCV2 Cap is the major component to suppress IL-12p40 expression in vivo. (A and B) The PAMs were infected by 100 MOI of rAd-Blank, rAd-Rep, or rAd-Cap for 24 h, respectively. Then the cells were stimulated by LPS/IFN-g or LPS/R848, and the protein and mRNA levels of IL-12p40 were measured by ELISA and qPCR. (C and D) The PAMs were infected by mock, PCV1, PCV2, PCV1-Rep2, PCV1-Cap2, (Figure legend continues) 8 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. gC1qR is critical for IL-12p40 suppression induced by PCV2 Cap protein. (A and B) PCV2 infected wild-type (gC1qR+/+) or gC1qR knockout (gC1qR2/2) PAMs for 24 h, and then the cells were stimulated by LPS/IFN-g for 6 or 24 h. The expression of IL-12p40 was measured by ELISA and qPCR. The expression of gC1qR were analyzed by Western blotting as the bottom panel shows. (C and D) The gC1qR+/+ and gC1qR2/2 PAMs were infected by rAd-Blank, rAd-Rep, or rAd-Cap for 24 h, respectively. Then the cells were further stimulated by LPS/IFN-g for 6 or 24 h, and the expression of IL-12p40 was measured by ELISA and qPCR. The expression of PCV2 Rep, Cap, and host gC1qR were analyzed as the bottom panel by Western blotting. **p , 0.01 versus PCV2- or rAd-Cap–infected gC1qR+/+ PAMs. ##p , 0.01 versus mock infection gC1qR+/+ PAMs. that miR-23a, miR-23b, miR-29a, and miR-29b mimics could with the specific inhibitors of miR-23a, miR-23b, miR-29a, and decrease the levels of relative luciferase activities from porcine miR-29b or inhibitor control, and then infected with PCV2 or mock il12B WT-39UTR compared with miRNA mimics control, but and stimulated by LPS/IFN-g. The inhibitors reduced the respective these miRNA mimics’ transfection did not affect the luciferase miRNA over 3.5-fold (Supplemental Fig. 3C). The specific inhib- activities of the cells transfected with the respective mutated-type itors of miR-23a and miR-29b could significantly reverse the reporters (Supplemental Fig. 3B). These data confirm that miR- PCV2-induced IL-12p40 suppression in protein levels, and miR- 23a, miR-23b, miR-29a, or miR-29b can transcriptionally regulate 29b could promote IL-12p40 transcription, whereas miR-23b– and the IL-12p40 expression via targeting its 39UTR. miR-29a–specific inhibitors did not significantly improve IL-12p40 Furthermore, to determine the regulatory roles of these miRNAs expression at either protein or mRNA level (Fig. 8C, 8D). Notably, in PCV2 inhibition of IL-12p40 expression, cells were transfected treatment with both miR-23a– and miR-29b–specific inhibitors

PCV2-Rep1, or PCV2-Cap1 at 1 MOI for 24 h, and then the cells were stimulated by LPS/IFN-g or LPS/R848 for another 24 h. The IL-12p40 secretion was 5 measured by ELISA. (E–H) Piglets were infected with mock, PCV1, PCV2, PCV1-Rep2, PCV1-Cap2, PCV2-Rep1, or PCV2-Cap1 at 4 3 10 TCID50 for 1 wk, and then the pigs were further infected by PRRSVor H. parasuis for 24 h. The serum IL-12p40 levels of the infected pigs and replication of PRRSV or H. parasuis were measured. (A and B)*p , 0.05, **p , 0.01 versus rAd-Blank–infected cells. #p , 0.05, ##p , 0.01 versus rAd-Rep–infected cells. (C–H)*p , 0.05, **p , 0.01 versus mock infection cells. #p , 0.05, ##p , 0.01 versus PCV1-infected cells. &p , 0.05, &&p , 0.01 versus PCV2- infected cells. $p , 0.05, $$p , 0.01 versus PCV1-Rep2– or PCV2-Cap1–infected cells. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. PCV2 infection activates PI3K/Akt1 and p38 MAPK signaling pathways to suppress IL-12p40 expression in transcriptional levels. The speciﬁc siRNAs of Akt1, p38 MAPK, ERK1, or negative control siRNA were transfected into PAMs for 24 h. Then the cells were infected by mock or PCV2 and followed by LPS/IFN-g stimulation. The IL-12p40 expression was detected by ﬂow cytometry (A) and qPCR (B). The binding activities of NF- kB p65 to il12B promoter were measured using chromatin immunoprecipitation assay (C). (D) The NF-kB p65 activity was measured by Dual-Luciferase reporter assays. *p , 0.05, **p , 0.01 versus negative control siRNA–transfected PAMs (si-NC) in same infection or mock. #p , 0.05, ##p , 0.01 versus control PAMs (CTRL) in same infection or mock. 10 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. PCV2 Cap upregulates miR-23a, miR-23b, miR-29a, and miR-29b expression in PAMs. (A) The PAMs were infected by PCV2 or mock for 24 h, and then the miRNAs that were predicted to regulate porcine IL-12p40 were analyzed by qPCR. (B) PCV2, PCV2-Cap1, PCV2-Rep1, or mock infected PAMs at 1 MOI for 24 h; the expressions of miR-23a, miR-23b, miR-29a, and miR-29b were analyzed by qPCR. (C) The rAd-Blank, rAd-Rep, and rAd-Cap infected PAMs at 100 MOI for 24 h; the expressions of miR-23a, miR-23b, miR-29a, and miR-29b were analyzed by qPCR. *p , 0.05, **p , 0.01 versus mock infection cells (A and B). #p , 0.05, ##p , 0.01 versus rAd-Blank–infected cells (C). could more significantly increase the LPS/IFN-g–induced IL-12p40 inhibitor control–transfected cells (Fig. 9A, 9B), suggesting that secretion in PCV2-infected PAMs than miR-23a or miR-29b in- reduction of miR-23a and miR-29b can improve the IL-12p40 ex- hibitor treatment alone and shows the same effect as that of the pression induced by other pathogens in PCV2-infected PAMs. Upon inhibitor mixture of four miRNAs (miR-23a, miR-23b, miR-29a, PRRSV or H. parasuis infection, the percentage of CD4+IFN-g+ and miR-29b) (Fig. 8C). These results suggest that miR-23a and Th1 cells was increased in the PBMC cocultured with anti–miR- miR-29b play a predominant role in PCV2 suppression of IL-12p40 23a– or anti–miR-29b–treated PAMs, particularly in PBMC cocultured expression. with anti–miR-mix–treated PAMs (Fig. 9C). In line with the increase of CD4+IFN-g+ Th1 cells, the levels of IFN-g in coculture Reduction of miR-23a and miR-29b induction improves system were markedly increased in cocultured cells with anti–miR- IL-12p40 expression of PAMs and Th1 immune and mix–treated PAMs (Fig. 9D). Consequently, the PRRSV replication represses the replication of other pathogens and H. parasuis replication were significantly decreased in the To confirm the roles of miR-23a and miR-29b during the IL-12p40 cocultured cells with anti–miR-mix–treated PAMs (Fig. 9E). These expression and host Th1 immune suppression by PCV2 infection, results further demonstrate the roles of miR-23a and miR-29b PAMs were transfected with miR-23a– and miR-29b–specific in- during PCV2 infection and that these two miRs are employed by hibitors (anti–miR-23a and anti–miR-29b) or inhibitor mix (anti- PCV2 to suppress IL-12p40 expression in PAMs, which results in a miR-mix), and then PAMs were infected by PCV2, incubated lower host Th1 immune response to other pathogens (Fig. 10). with PBMC isolated from healthy piglets, and challenged with PRRSV or H. parasuis. Upon PRRSV or H. parasuis challenge, Discussion IL-12p40–positive cell percentage and expression were increased in Swine are one of the major animal species used in medical research either anti–miR-23a– or anti–miR-29b–treated PAMs, particularly because they are very similar to humans in terms of genetics, in PAMs treated with anti–miR-mix, when compared with the miR anatomy, physiology, and immunology (23, 24). Aside from the The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 7. PCV2 Cap and gC1qR interaction activates PI3K/Akt1 and p38 MAPK signaling to regulate miR-23a, miR-23b, miR-29a, and miR-29b expression. (A) PCV2 infected gC1qR+/+ and gC1qR2/2 PAMs at 1 MOI for 24 h; the expressions of miR-23a, miR-23b, miR-29a, and miR-29b were by guest on September 27, 2021 measured. (B–D) The specific siRNAs of Akt1 (B), p38 MAPK (C), and ERK1 (D) were transfected into wild-type PAMs for 24 h, and then the cells were infected with 1 MOI of PCV2 for another 24 h. The expressions of miR-23a, miR-23b, miR-29a, and miR-29b were analyzed. *p , 0.05, **p , 0.01 versus PCV2-infected gC1qR+/+ PAMs for same detected miRNA (A). #p , 0.05, ##p , 0.01 versus PCV2-infected negative control siRNA–transfected cells for same miRNA detected (B–D). primate and murine immune systems, the swine immune system is induction of host Th1 cell immune response (28). Several viruses probably the best characterized one (24). Like other mammals, have been shown to suppress host IL-12 expression. For example, pigs have a full set of innate and adaptive immune effectors, most HIV type 1–positive subjects show a significant decrease in levels of which share structural and functional similarities with their of IL-12 (29). Chronic hepatitis B virus infection is found to se- human counterparts (25). All these specialties make pigs an lectively inhibit TLR2 ligand–induced IL-12p40 mRNA expres- excellent animal model for studying the immunopathological sion in PMA-differentiated THP-1 macrophages (30). PRRSV mechanisms of various infectious diseases. Previous works have infection reduces IL-12p40 secretion in monocyte-derived den- demonstrated that PCV2 infection can suppress the host immune dritic cells (31). Among the negative regulators of IL-12 expres- system, leading to the PCV2-infected pigs’ coinfection with other sion, IL-10 plays an important role as an anti-inflammatory pathogens (2). In contrast, PCV1 infection does not induce path- cytokine (22). In chronic hepatitis C virus patients, IL-10 is found ological changes (26). In this study, we investigated how PCV2 to suppress TLR4 agonist–induced IL-12 production (32). PCV2 infection interferes with the immune response to other pathogens. infection has been shown to upregulate IL-10 production in PAMs The results demonstrate that PCV2 infection significantly sup- during the 24 h after PCV2 infection in a previous study (19). presses other pathogen- or PAMP molecule–induced IL-12p40 Although IL-10 might be also involved in negative regulation of expression of PAMs, leading to a relatively lower Th1 immune IL-12 expression in PCV2-infected cells, IL-10 is not required for response and a weakened pathogenic clearance upon other path- PCV2 suppression of IL-12p40 induction. In the IL-10–deficient ogen infections in PCV2-infected piglets than in PCV1-infected macrophages, PCV2 infection also inhibited IL-12p40 expression piglets. In PCV2-infected PAMs, PCV2 Cap interaction with (Supplemental Fig. 3D), suggesting that PCV2 suppression of gC1qR activates PI3K/Akt1 and p38 MAPK signaling to inhibit IL-12p40 induction is not completely dependent on IL-10. PCV2 NF-kB transcriptional activity and upregulates miR-23a and miR- is also reported to suppress CpG- or R837-induced IL-12p40 ex- 29b to suppress IL-12p40 expression at both transcriptional and pression in dendritic cells (33), but the mechanism has been un- posttranscriptional levels (Fig. 10). known until this work. Macrophages are major innate immune cells involved in In a previous study, Cap protein was identified as a critical detecting infections, anti-infection response, and inflammation player in induction of IL-10 by PCV2 (19). In this work, rAd- aiming to pathogen elimination (20, 27). IL-12 is the key proin- RepandrAd-CapshowedthatbothRepandCapcouldinhibit flammatory cytokine produced by activated macrophages in the IL-12p40 expression induced by LPS/IFN-g or LPS/R848 12 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs Downloaded from http://www.jimmunol.org/

FIGURE 8. miR-23a and miR-29b play critical roles at posttranscriptional suppression of IL-12p40 expression in PCV2-infected cells. (A and B) The miR-23a, miR-23b, miR-29a, and miR-29b mimics were transfected into PAMs for 24 h, and then the cells were stimulated by LPS/IFN-g or LPS/R848 for by guest on September 27, 2021 another 6 or 24 h. The expression of IL-12p40 was measured by ELISA and qPCR. (C and D) The specific inhibitors of miR-23a, miR-23b, miR-29a, and miR-29b were transfected into PAMs separately or combined, and then the cells were infected by 1 MOI PCV2 for 24 h. The cells were further stimulated by LPS/IFN-g for 6 or 24 h. The expression of IL-12p40 was measured by ELISA and qPCR. (A and B)*p , 0.05, **p , 0.01 versus negative control mimic–transfected cells. (C and D)*p , 0.05, **p , 0.01 versus negative control inhibitor–transfected PCV2-infected cells. #p , 0.05, ##p , 0.01 versus the PCV2-infected cells transfected with the mixture of all four miRNA inhibitors. stimulation. However, PCV mutants containing PCV2 Rep evidence that LPS/IFN-g induced more IL-12p40 in the gC1qR without PCV2 Cap (PCV1-Rep2 and PCV2-Cap1) did not ex- knockout PAMs than in the wild-type PAMs when the cells were hibit a significantly inhibitory effect on IL-12p40 expression infected by PCV2 or rAd-Cap. These data demonstrate that the in vivo or in vitro. Because PCV2 replication is very slow or interaction of Cap with gC1qR plays important roles in both IL-10 limited in macrophages, Rep expression is extremely low in the and IL-12p40 regulation at different times after PCV2 infection. PCV1-Rep2– or PCV2-Cap1–infected PAMs. Thus, we specu- IL-12p40 expression is regulated by multiple signaling pathways lated that the difference of Rep in IL-12p40 suppression be- in macrophages (11, 28). Previous studies have shown that PCV2 tween rAd-Rep and PCV mutants might be due to the level of infection activates PI3K/Akt, p38 MAPK, and ERK pathways Rep protein in the cells. Massively exogenous overexpression of through Cap and gC1qR interaction (19). All of these pathways Rep is able to suppress IL-12p40 induction in PAMs infected have been reported to participate in the regulation of IL-12p40 with rAd-Rep, but PCV mutants contain PCV2 Rep, which is expression (22). It has been reported that gC1qR ligation selec- not able to suppress IL-12p40 induction because of a lower Rep tively inhibits TLR4-induced IL-12 production through activation levelthatisbarelydetectedincells. These results indicate that of the PI3K pathway in human macrophages (37). Inhibition of Cap is a more critical compound in induction of IL-12p40 Akt enhances IL-12 production in Giardia lamblia trophozoite– suppression relative to Rep during PCV2 infection. stimulated mouse peritoneal macrophages (38). p38 MAPK me- Our previous study has also found that PCV2 Cap binds with diates IL-12p40 inhibition by NO in macrophages (39). ERK is gC1qR to regulate IL-10 production in PAMs (19). gC1qR is a activated to inhibit IL-12 production in macrophages treated with major host protein that has been exploited by a wide range of Leishmania lipophosphoglycans (40). Furthermore, research also bacterial and viral ligands to suppress the host’s immune response found that PI3K/Akt is required for Neospora-induced p38 MAPK to promote their survival (34). To date, gC1qR has been reported downregulation of IL-12 production (41). In this study, inhibition as interacting with the hepatitis C virus core protein to suppress of either Akt1 or p38 MAPK by specific siRNAs reversed the the IL-12 synthesis in macrophages and dendritic cells (35, 36). In PCV2-induced IL-12p40 suppression, whereas silence of ERK1 this study, gC1qR was also found to play a crucial role in the did not alter the IL-12p40 suppression. Inhibition of Akt1 or p38 suppression of IL-12p40 expression, which was supported by the MAPK enhanced IL-12p40 expression in PAMs upon LPS/IFN-g The Journal of Immunology 13 Downloaded from

FIGURE 9. Reduction of miR-23a and miR-29b improves IL-12p40 expression and Th1 immune response and suppresses the replication of other pathogens. The PAMs were transfected with miR-23a– and miR-29b–specific inhibitors and then http://www.jimmunol.org/ infected with PCV2 or mock at 1 MOI for 24 h. Then PAMs were incubated with PBMC isolated from healthy piglets in a 1:1 ratio and further challenged with PRRSV or H. parasuis for 24 h. (A and B) The IL-12p40 expression of the PAMs was analyzed by flow cytometry and ELISA. (C and D) The percentage of IFN- g+CD4+ T cells was analyzed by flow by guest on September 27, 2021 cytometry, and the levels of IFN-g in coculture system were measured by ELISA. (E) The replication of PRRSV or H. parasuis was measured. *p , 0.05, **p , 0.01 versus PCV2 alone or PCV2 plus PRRSV or H. parasuis infection without specific miRNA inhibitor.

stimulation. Inhibition of Akt1 induced signiﬁcantly higher were considered upstream signaling that promotes NF-kB activi- IL-12p40 expression than inhibition of p38 MAPK in PCV2- ties (42–44). But, recently, other studies show that PI3K/Akt1 and infected PAMs. Previously, PI3K/Akt and p38 MAPK pathways p38 MAPK can inhibit NF-kB p65 activities in macrophages, that 14 PCV2 SUPPRESSES IL-12p40 INDUCTION IN PAMs Downloaded from

FIGURE 10. Model of PCV2 infection inhibits IL-12p40 expression in PAMs to further suppress host Th1 immune response. PCV2 infection activates http://www.jimmunol.org/ PI3K/Akt1 and p38 MAPK signaling pathways via Cap and gC1qR interaction to suppress other pathogen- or PAMP molecule–induced IL-12p40 expression at transcriptional levels. The activated PI3K/Akt1 and p38 MAPK pathways also induce miR-23a and miR-29b upregulation to suppress IL-12p40 expression at transcriptional and posttranscriptional levels. The reduction of IL-12 secretion by PAMs results in a lower host Th1 immune response to pathogen infection. The green arrows mean induction, the red lines mean inhibition, and the red arrows mean downregulation.

PI3K/Akt1 contributes to M2 polarization and negative regulates regulating miR-29a and miR-29b expression in PCV2-infected NF-kB p65 signaling to inhibit IL-12 expression (37, 45, 46), and PAMs. Because the inhibition of Akt1 and p38 MAPK did not that p38 MAPK has been employed by some pathogens to sup- totally inhibit the miRNA expression, we considered Akt1 and p38 press IL-12 production (41, 47, 48). These results suggest that the participators in the regulation of miRNAs, but they were not the by guest on September 27, 2021 roles of Akt1 and p38 MAPK are alterative in the regulation of only regulators. Among four miRNAs, miR-23a and miR-29b NF-kB p65 transcriptional activity during different infections, were markedly upregulated by PCV2 and played a predominant during different stages of infection, or in different cells. In the role in regulation of IL-12p40 at posttranscriptional level. Inhi- current study, Akt1 and p38 MAPK signaling was also confirmed bition of both miR-23a and miR-29b could reverse the IL-12p40 to inhibit the NF-kB p65 transcriptional activity and binding ac- expression, as long as all four miRNA inhibitors work together. tivities of NF-kB p65 to IL-12p40 promoter in PCV2-infected Because the PCV2 infection also suppressed IL-12p40 expression PAMs. However, more detailed information about how PI3K/ at transcriptional level, the suppression of only posttranscriptional Akt1 and p38 MAPK signal inhibits NF-kB transcriptional ac- levels could not completely reverse the IL-12p40 expression. tivity is continuously studied. Interestingly, the data presented in Because it is hard to confirm the action of miRNAs in vivo, we this paper suggest that p38 MAPK negatively regulates IL-12p40 used a coculture experiment to determine the action of these expression in PAMs, particularly in the cells that AKT1 signaling miRNAs in PCV2-induced IL-12p40 suppression. We found that activated at the same time. reduction of miR-23a and miR-29b induction in the PCV2- In addition, we observed that the reduction of IL-12p40 mRNA infected PAMs could improve IL-12p40 expression and promote levels was not as great as that of protein levels. Based on that, we cocultured PBMCs to produce more IFN-g–positive T cells. further screened and identified the miRNAs that could inhibit IL- Meanwhile, the replication of challenged PRRSV or H. parasuis 12p40 expression of PAMs at posttranscriptional level. Several was significantly inhibited. In this case, we figured out that PCV2 miRNAs have been reported to regulate human or mouse IL-12p40 infection suppresses IL-12p40 expression at posttranscriptional expression at posttranscriptional level (28). miR-23 is the only levels, mainly via upregulation of miR-23a and miR-29b in PAMs. broadly conserved miRNA family among vertebrates with a In summary, this study provides certain evidence that PCV2 conserved site in IL-12p40 39UTR, based on TargetScan and infection suppresses other pathogen-induced IL-12p40 expression miRWalk. Aside from that, several poorly conserved sites for other at both transcriptional and posttranscriptional levels through the conserved miRNA families can be found on porcine IL-12p40 Cap and gC1qR interaction–mediated PI3K/Akt1 and p38 MAPK 39UTR. In PCV2-infected PAMs, miR-23a, miR-23b, miR-29a, pathway activation and miR-23a and miR-29b upregulation, and miR-29b were found to be upregulated. These miRNAs resulting in a lower Th1 immune response and a weakened were confirmed to be directly binding to the 39UTR of IL-12p40 pathogenic clearance. Inhibition of PI3K/Akt1 and p38 MAPK mRNA and negatively regulated IL-12p40 expression. The up- pathway activation and downregulation of miR-23a and miR-29b regulation of all of these four miRNAs were dependent on the can decrease the risk of secondary infection in PCV2-infected presence of Cap binding protein gC1qR. In the signaling activated animals. These findings might help us to further understand the by PCV2, PI3K/Akt1 was involved in the regulation of miR-23a, relative immune mechanisms determining the susceptibility of miR-23b, and miR-29b expression; p38 MAPK was involved in PCV2-infected animals. The Journal of Immunology 15

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