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

Affiliations
  • 1College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. lyj@njau.edu.cn

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.


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