J Vet Sci.  2017 Jun;18(2):183-191. 10.4142/jvs.2017.18.2.183.

Porcine circovirus type 2 increases interleukin-1beta and interleukin-10 production via the MyD88–NF-kappa B signaling pathway in porcine alveolar macrophages in vitro

• Han J
• Zhang S
• Zhang Y
• Chen M
• Lv Y

• KMID: 2412571
• DOI: http://doi.org/10.4142/jvs.2017.18.2.183

Abstract

Porcine alveolar macrophages (PAMs) represent the first line of defense in the porcine lung after infection with porcine circovirus type 2 (PCV2) via the respiratory tract. However, PCV2 infection impairs the microbicidal capability of PAMs and alters cytokine production and/or secretion. At present, the reason for the imbalance of cytokines has not been fully elucidated, and the regulatory mechanisms involved are unclear. In this study, we investigated the expression levels and regulation of interleukin-1beta (IL-1β) and IL-10 in PAMs following incubation with PCV2 in vitro. Levels of IL-1β and IL-10 increased in PAM supernatants, and the distribution of nuclear factor kappa B (NF-κB) p65 staining in nucleus, expression of MyD88 and p-IκB in cytoplasm, and DNA-binding activity of NF-κB increased after incubation with PCV2, while p65 expression in PAM cytoplasm decreased. However, when PAMs were co-incubated with PCV2 and small interfering RNA targeting MyD88, those effects were reversed. Additionally, mRNA expression levels of Toll-like receptors (TLR)-2, -3, -4, -7, -8, and -9 increased when PAMs were incubated with PCV2. These results show that PCV2 induces increased IL-1β and IL-10 production in PAMs, and these changes in expression are related to the TLR-MyD88-NF-κB signaling pathway.

Keyword

NF-kappa B; interleukin-10; interleukin-1beta; porcine alveolar macrophage; porcine circovirus type 2

MeSH Terms

Animals
Circoviridae Infections/metabolism/*veterinary/virology
*Circovirus/metabolism
In Vitro Techniques
Interleukin-10/*metabolism
Interleukin-1beta/*metabolism
Macrophages, Alveolar/*metabolism/virology
Myeloid Differentiation Factor 88/*physiology
NF-kappa B/*physiology
*Signal Transduction/physiology
Swine
Swine Diseases/metabolism/virology
Interleukin-1beta
Myeloid Differentiation Factor 88
NF-kappa B
Interleukin-10

Figure

• 

  Fig. 1 Porcine circovirus type 2 (PCV2) infection in piglet porcine alveolar macrophages (PAMs). Control PAM cultures (A) and PAMs incubated with PCV2 for 6, 12, 24, and 48 h (B–E, respectively) were stained with anti-PCV2 antibody (green) and visualized by indirect immunofluorescence. DAPI was used to stain nuclei (blue).

• 

  Fig. 2 Changes in interleukin-1beta (IL-1β) and IL-10 levels after porcine circovirus type 2 (PCV2) infection. Supernatants from control cultures and porcine alveolar macrophages cultured with PCV2, MyD88 small interfering (si)RNA, or a combination of both were sampled after 6, 12, 24, and 48 h and were underwent enzyme-linked immunosorbent assay to measure the concentrations of IL-1β and IL-10; *p < 0.05, **>p < 0.01.

• 

  Fig. 3 Changes in MyD88 protein expression after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure the expression of MyD88 protein in the cytoplasm at 6, 12, 24, and 48 h in control, PCV2, MyD88 siRNA, and PCV2 +MyD88 small interfering (si)RNA groups. Expression of β-actin was used as a positive control; *p < 0.05, **>p < 0.01.

• 

  Fig. 4 Localization of nuclear factor kappa B p65 in the nucleus of porcine alveolar macrophages (PAMs) after porcine circovirus type 2 (PCV2) infection for 24 h. Control PAM cultures (A) and PAMs incubated with PCV2, MyD88 small interfering (si)RNA, and PCV2 + MyD88 siRNA (B–D, respectively) for 24 h were stained with a mouse monoclonal antibody against p65 (red) and visualized by indirect immunofluorescence. DAPI was used to stain nuclei (blue).

• 

  Fig. 5 Changes in nuclear factor kappa B (NF-κB) p65 protein expression in the cytoplasm after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure cytoplasmic expression of NF-κB p65 at 6, 12, 24, and 48 h in the control, PCV2, MyD88 small interfering (si)RNA, and PCV2 + MyD88 siRNA groups. Expression of β-actin was used as a positive control; *p < 0.05, **p < 0.01.

• 

  Fig. 6 Changes in nuclear factor KappaB inhibitor alpha (p-IκBα) protein expression after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure the cytoplasmic expression of p-IκBα protein at 6, 12, 24, and 48 h in the control, PCV2, MyD88 small interfering (si)RNA, and PCV2 + MyD88 siRNA groups. Expression of GADPH was used as a positive control; *p < 0.05, **p < 0.01.

• 

  Fig. 7 Changes in nuclear factor kappa B (NF-κB) DNA-binding activity after porcine circovirus type 2 (PCV2) infection. An electrophoretic mobility shift assay was used to measure NF-κB DNA-binding activity after 6, 12, 24, and 48 h in the control, PCV2, MyD88 small interfering (si)RNA, and PVC2 + MyD88 siRNA groups; *p < 0.05, **p < 0.01.

• 

  Fig. 8 Changes in mRNA expression levels of various Toll-like receptors (TLRs) after porcine circovirus type 2 (PCV2) infection. Real-time RT-PCR was used to assess the mRNA expression levels of TLR-2, -3, -4, -7, -8, and -9 in porcine alveolar macrophages incubated with PCV2 and controls after 6, 12, 24, and 48 h; *p 0.05, **p < 0.01.

Reference

1. Allan GM, Ellis JA. Porcine circoviruses: a review. J Vet Diagn Invest. 2000; 12:3–14.

2. Chae JS, Choi KS. Proinflammatory cytokine expression in the lung of pigs with porcine circovirus type 2-associated respiratory disease. Res Vet Sci. 2011; 90:321–323.

3. Chang HW, Jeng CR, Lin TL, Liu JJ, Chiou MT, Tsai YC, Chia MY, Jan TR, Pang VF. Immunopathological effects of porcine circovirus type 2 (PCV2) on swine alvelolar macrophages by in vitro inoculation. Vet Immunol Immunopathol. 2006; 110:207–219.

4. Chang HW, Pang VF, Chen LJ, Chia MY, Tsai YC, Jeng CR. Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages. Vet Microbiol. 2006; 115:311–319.

5. Darwich L, Pié S, Rovira A, Segalés J, Domingo M, Oswald IP, Mateu E. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisytemic wasting syndrome. J Gen Virol. 2003; 84:2117–2125.

6. Darwich L, Segalés J, Mateu E. Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: an immune riddle. Arch Virol. 2004; 149:857–874.

7. Duan D, Zhang S, Li X, Guo H, Chen M, Zhang Y, Han J, Lv Y. Activation of the TLR/MyD88/NF-κB signal pathway contributes to changes in IL-4 and IL-12 production in piglet lymphocytes infected with porcine circovirus type 2 in vitro. PloS One. 2014; 9:e97653.

8. Ellis JA, Allan G, Krakowka S. Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs. Am J Vet Res. 2008; 69:1608–1614.

9. Gilpin DF, McCullough K, Meehan BM, McNeilly F, McNair I, Stevenson LS, Foster JC, Ellis JA, Krakowka S, Adair BM, Allan GM. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system. Vet Immunol Immunopathol. 2003; 94:149–161.

10. Hertzog PJ, O’Neill LA, Hamilton JA. The interferon in TLR signaling: more than just antiviral. Trends Immunol. 2003; 24:534–539.

11. Kekarainen T, Montoya M, Mateu E, Segalés J. Porcine circovirus type 2-induced interleukin-10 modulates recall antigen responses. J Gen Virol. 2008; 89:760–765.

12. Krakowka S, Ellis J, McNeilly F, Waldner C, Rings DM, Allan G. Mycoplasma hyopneumoniae bacterins and porcine circovirus type 2 (PCV2) infection: induction of postweaning multisystemic wasting syndrome (PMWS) in the gnotobiotic swine model of PCV2-associated disease. Can Vet J. 2007; 48:716–724.

13. Li J, Yu Q, Nie X, Guo X, Song Q, Li H. Effects of porcine circovirus type 2 on expression of mRNA associated with endogenous antigen processing and presentation in pulmonary alveolar macrophages and circulating T lymphocytes in piglets. Vet J. 2012; 193:199–205.

14. Liu J, Bai J, Lu Q, Zhang L, Jiang Z, Michal JJ, He Q, Jiang P. Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2. J Proteomics. 2013; 79:72–86.

15. Lv Y, Dai L, Han H, Zhang S. PCV2 induces apoptosis and modulates calcium homeostasis in piglet lymphocytes in vitro. Res Vet Sci. 2012; 93:1525–1530.

16. Lv Y, Zhang X, Sun Y, Zhang S. Activation of NF-κB contributes to production of pig-major acute protein and serum amyloid A in pigs experimentally infected with porcine circovirus type 2. Res Vet Sci. 2013; 95:1235–1240.

17. Mandrioli L, Sarli G, Panarese S, Baldoni S, Marcato PS. Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol. 2004; 97:25–37.

18. Mosser DM, Zhang X. Interleukin-10: new perspectives on an old cytokine. Immunol Rev. 2008; 226:205–218.

19. Pang VF, Lambert RJ, Felsburg PJ, Beasley VR, Buck WB, Haschek WM. Experimental T-2 toxicosis in swine following inhalation exposure: effects on pulmonary and systemic immunity, and morphological changes. Toxicol Pathol. 1987; 15:308–319.

20. Rovira A, Balasch M, Segalés J, García L, Plana-Durán J, Rosell C, Ellerbrok H, Mankertz A, Domingo M. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. J Virol. 2002; 76:3232–3239.

21. Sabat R. IL-10 family of cytokines. Cytokine Growth Factor Rev. 2010; 21:315–324.

22. Shi KC, Guo X, Ge XN, Liu Q, Yang HC. Cytokine mRNA expression profiles in peripheral blood mononuclear cells from piglets experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2. Vet Microbiol. 2010; 140:155–160.

23. Wei L, Kwang J, Wang J, Shi L, Yang B, Li Y, Liu J. Porcine circovirus type 2 induces the activation of nuclear factors kappa B by IκBα degradation. Virology. 2008; 378:177–184.

24. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi Q, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science. 2003; 301:640–643.

25. Yu S, Opriessnig T, Kitikoon P, Nilubol D, Halbur PG, Thacker E. Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs. Vet Immunol Immunopathol. 2007; 115:261–272.