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 Supplementary http://www.jimmunol.org/content/suppl/2018/05/31/jimmunol.180025 http://www.jimmunol.org/ Material 0.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 sup- pressed 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.