Non-infectious immune complexes downregulate the production of interferons and tumor necrosis factor-α in primary porcine alveolar macrophages in vitro

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) has been harming the pig industry worldwide for nearly 40 years. Although scientific researchers have made substantial efforts to explore PRRSV pathogenesis, the immune factors influencing PRRSV infection still need to be better understood. Infectious virus-antibody immune complexes (ICs) formed by PRRSV and sub-or non-neutralizing antibodies specific for PRRSV may significantly promote the development of PRRS by enhancing PRRSV replication through antibody-dependent enhancement. However, nothing is known about whether PRRSV infection is affected by non-infectious ICs (NICs) formed by non-pathogenic/infectious antigens and corresponding specific antibodies. Here, we found that PRRSV significantly induced the transcripts and proteins of interferon-α (IFN-α), IFN-β, IFN-γ, IFN-λ1, and tumor necrosis factor-α (TNF-α) in vitro primary porcine alveolar macrophages (PAMs) in the early stage of infection. Our results showed that NICs formed by rabbit-negative IgG (RNI) and pig anti-RNI specific IgG significantly reduced the transcripts and proteins of IFN-α, IFN-β, IFN-γ, IFN-λ1, and TNF-α in vitro PAMs and significantly elevated the transcripts and proteins of interleukine-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in vitro PAMs. NICs-mediated PRRSV infection showed that NICs not only significantly decreased the induction of IFN-α, IFN-β, IFN-γ, IFN-λ1, and TNF-α by PRRSV but also significantly increased the induction of IL-10 and TGF-β1 by PRRSV and considerably enhanced PRRSV replication in vitro PAMs. Our data suggested that NICs could downregulate the production of antiviral cytokines (IFN-α/β/γ/λ1 and TNF-α) during PRRSV infection in vitro and facilitated PRRSV proliferation in its host cells by inhibiting innate antiviral immune response. This study elucidated one novel immune response to PRRSV infection, which would enhance our understanding of the pathogenesis of PRRSV.

Keywords: IFNs; NICS; PAMS; PRRSV; TNF-α.

Figure 1

Effect of PRRSV or NICs on immune cytokine mRNAs in PAMs. PAM cell monolayers were incubated with PRRSV (200 TCID₅₀), PNI + RNI, NICs, or LPS (100 ng/mL). Cells were collected for total RNA isolation at 12, 24, 36, 48, 60, or 72 h later. IFN-α (A), IFN-β (B), IFN-γ (C), IFN-λ1 (D), TNF-α (E), IL-10 (F), or TGF-β1 (G) mRNA expression was analyzed by relative quantitative RT-PCR method. β-actin was used to normalize cytokine mRNA levels. ^***p < 0.001, ^**p < 0.01, ^*p < 0.05. Bars indicate the 2^−∆CT of mRNA copies of cytokines in cells ± SEM from three repeated experiments. LPS, lipopolysaccharide; NICs, non-infectious immune complexes; PNI, pig-negative IgG; RNI, rabbit-negative IgG; ns, no significance.

Figure 2

Effect of PRRSV or NICs on immune cytokine proteins in PAMs. PAM cell monolayers were incubated with PRRSV (200 TCID₅₀), PNI + RNI, NICs, or LPS (100 ng/mL). Cell supernatants were collected for ELISA assay at 12, 24, 36, 48, 60, or 72 h later. IFN-α (A), IFN-β (B), IFN-γ (C), IFN-λ1 (D), TNF-α (E), IL-10 (F), or TGF-β1 (G) protein production was quantified using commercial ELISA kits. ^***p < 0.001, ^**p < 0.01, ^*p < 0.05. Bars indicate the mean of protein concentrations of cytokines in cell supernatants ± SEM from three repeated experiments. LPS, lipopolysaccharide; NICs, non-infectious immune complexes; PNI, pig-negative IgG; RNI, rabbit-negative IgG; ns: no significance.

Figure 3

The kinetics of PRRSV proliferation in PAMs. PAM cell monolayers were infected with 200 TCID₅₀ of PRRSV. Cell supernatants were harvested at 12, 24, 36, 48, 60, or 72 h post-infection. PRRSV RNA copies (A) and its TCID₅₀/mL titers (B) in cell supernatants were measured by real-time RT-PCR and TCID₅₀ assay. Data represents mean ± SEM of three repeated experiments.

Figure 4

Effect of NICs on immune cytokine mRNAs in PAMs infected with PRRSV. PAM cell monolayers were pretreated with PNI + RNI or NICs for 2 h and then infected with 200 TCID₅₀ of PRRSV. Cells were collected for total RNA isolation at 12, 24, 36, 48, 60, or 72 h post-infection. IFN-α (A), IFN-β (B), IFN-γ (C), IFN-λ1 (D), TNF-α (E), IL-10 (F), or TGF-β1 (G) mRNA expression was analyzed by relative quantitative RT-PCR method. β-actin was used to normalize cytokine mRNA levels. ^***p < 0.001, ^**p < 0.01, ^*p < 0.05. Bars indicate the 2^−∆CT of mRNA copies of cytokines in cells ± SEM from three repeated experiments. NICs, non-infectious immune complexes; PNI, pig-negative IgG; RNI, rabbit-negative IgG.

Figure 5

Effect of NICs on immune cytokine proteins in PAMs infected with PRRSV. PAM cell monolayers were pretreated with PNI + RNI or NICs for 2 h and then infected with 200 TCID₅₀ of PRRSV. Cell supernatants were collected for ELISA assay at 12, 24, 36, 48, 60, or 72 h post-infection. IFN-α (A), IFN-β (B), IFN-γ (C), IFN-λ1 (D), TNF-α (E), IL-10 (F), or TGF-β1 (G) protein production was quantified using commercial ELISA kits. ^***p < 0.001, ^**p < 0.01, ^*p < 0.05. Bars indicate the mean of protein concentrations of cytokines in cell supernatants ± SEM from three repeated experiments. NICs, non-infectious immune complexes; PNI, pig-negative IgG; RNI, rabbit-negative IgG.

Figure 6

Effect of NICs on PRRSV propagation in PAMs. PAM cell monolayers were pretreated with PNI + RNI or NICs for 2 h and then infected with 200 TCID₅₀ of PRRSV. Cell supernatants were harvested at 12, 24, 36, 48, 60, or 72 h post-infection. PRRSV RNA copies (A) and its TCID₅₀/mL titers (B) in cell supernatants were measured by real-time RT-PCR and TCID₅₀ assay. ^***p < 0.001. Bars indicate RNA copies or TCID₅₀/mL titers of PRRSV. Error bars indicate the mean ± SEM of three repeated experiments. NICs, non-infectious immune complexes; PNI, pig-negative IgG; RNI, rabbit-negative IgG.

Figure 7

Immunoblot analysis of PRRSV N protein in PAMs. PAM cell monolayers were pretreated with PNI + RNI or NICs for 2 h and then infected with 200 TCID₅₀ of PRRSV. Cells were collected for protein extraction at 12, 24, 36, 48, 60, or 72 h post-infection. Whole-cell proteins were prepared for western blotting with primary (rabbit anti-PRRSV-N protein pAb, 1:1000 dilution) and secondary antibodies (goat anti-rabbit IgG-HRP antibody, 1:10000 dilution). β-actin served as a loading control. NICs, non-infectious immune complexes; PNI, pig-negative IgG; RNI, rabbit-negative IgG; pAb, polyclonal antibody.