AIF1 was identified as an up-regulated gene contributing to CSFV Shimen infection in porcine alveolar macrophage 3D4/21 cells

Study design, virus strain, and cells

DGE analysis was performed to screen for differentially expressed genes in CSFV-infected PAMs based on previously reported high-throughput sequencing data (Ning et al., 2017), which identified AIF1 as a key gene, and further testing was conducted to confirm its role during infection by CSFV Shimen. The CSFV Shimen strain was obtained from the Control Institute of Veterinary Bioproducts and Pharmaceuticals. The PAM 3D4/21 cell line (American Type Culture Collection: CRL-2843) were seeded at a density of 2 × 10⁷ cells per flask. When the macrophages reached 70–80% confluence, CSFV Shimen was added to the cultures at a multiplicity of infection of 5 TCID50 (Ning et al., 2017). After incubation with the virus at 37 °C and 5% CO₂ for 1 h, the virus was removed, and the cells were cultured for 0, 12, 24, and 48 h, and RNA or protein extracts from the cultured cells were collected for subsequent detection (Ning et al., 2016).

Quantitative polymerase chain reaction

To assess the differences in mRNA expression by quantitative polymerase chain reaction (qPCR), total mRNA was extracted from CSFV-infected and uninfected PAMs. Then, complementary DNA (cDNA) was synthesized from the mRNA by using the Transcriptor First Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Finally, changes in the mRNA expression of AIF1, IL6, and β-actin in the experimental groups (CSFV-infected PAMs) were detected by qPCR conducted on an ABI7300 system using SuperReal PreMix Plus (SYBR Green) and the specific oligonucleotide primers shown in Table 1. The two-step thermal cycler program was as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 63 °C for 30 s. Three replicate experiments were performed for each experimental group, and the Ct value for each sample was calculated and normalized using the 2^−ΔΔCt method.

Western blotting

Total protein was extracted from each cell sample, and the protein concentration was measured using the BCA protein assay kit (Cowin Biotech). Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline and Tween 20 (TBST) for 1 h at 25 °C ± 2 °C, and then incubated with specific primary antibodies raised against AIF1 (ab5076; Abcam), IL6 (ab6672; Abcam), CSFV E2 (MssBio, China), and β-actin (Tianjin Sungene Biotech, China). The membranes were washed with TBST solution six times for 5 min each, and then washed in the same manner after incubation with the secondary antibody for 1 h at room temperature. Western blotting images were obtained using a GeneGnome XRQ chemiluminescence detector (Syngene) and quantified using ImageJ software (National Institutes of Health).

Confocal immunofluorescence microscopy

PAMs infected with CSFV were washed with phosphate-buffered saline (PBS), fixed with acetone/methanol (1:1) at room temperature for 20 min, and permeabilized with 1% Triton X-100 in PBS for approximately 10 min. The samples were washed with PBS three times, and then incubated with a mouse anti-E2 antibody and a rabbit anti-AIF1 or anti-IL6 antibody at room temperature for 1 h, and then incubated with donkey anti-rabbit IgG and donkey anti-mouse IgG for 1 h at room temperature. Finally, the samples were incubated with 4′, 6-diamidino-2-phenylindole (DAPI), and examined using a confocal microscope (LSM510 META, Zeiss, Germany). Image analysis was performed using the operating software provided with the microscope.

Gene overexpression and RNA interference

A recombinant plasmid overexpressing AIF1 was constructed, and the primers including restriction sites (Table 1) were designed to amplify AIF1 gene according to archived AIF1 nucleotide sequence. The product of PCR was subcloned into mammalian expression vector pEGFP-C1 (YouBio, China). The constructed AIF1 overexpressed plasmids (p-AIF1) and empty plasmids (p-AIF1-NC) were introduced into overnight cultured macrophages in 6-well plates at 37 °C by using turbofect transfection reagent (Thermo Scientific, Waltham, MA, USA). At 12 h post-transfection, cells were treated with different research requirements.

Three pairs of shRNA sequences targeting different nucleotide sites in the porcine AIF1 gene were designed by using Invitrogen siRNA online design software (https://rnaidesigner.invitrogen.com/rnaiexpress). A BLAST search was performed to ensure that the shRNAs did not have significant sequence homology with other genes. The 63-nt oligonucleotides were annealed and cloned into the BamHI and HindIII sites of the pGenesil-1 vector (Yingrun Biotech. Co., Ltd., China). The shRNA expression vectors were named AIF1-p-Si1, AIF1-p-Si2, and AIF1-p-Si3. A scrambled negative shRNA, which was named AIF1-p-SiNC, was used as a control. All constructs were confirmed by DNA sequencing using the Sanger method, and are listed in Table 1. TurboFect™ transfection reagent was used to transfect AIF1-p-Si1, AIF1-p-Si2, AIF1-p-Si3, and AIF1-p-SiNC into 3D4/21 cells in 6-well plates at 37 °C and 5% CO₂. After 16–24 h of transfection, fluorescence microscopy was used to observe the effects of shRNA transfection.

Statistical analysis

Statistical analysis was carried out by one-way ANOVA using IBM SPSS software (https://www.ibm.com/analytics), and P values less than 0.05 were considered statistically significant. Experimental data are reported as mean values ± SE (n = 3).

Upregulation of AIF1 and IL6 transcription following infection by CSFV Shimen

After the PAMs 3D4/21 cells were infected with CSFV-Shimen for 48 h, the DGE data showed that AIF1 expression was higher in infected cells than in the control PAMs (Fig. 1A), indicating that CSFV Shimen infection upregulates the transcription level of AIF1. Analysis of the basic characteristics of porcine AIF1 is shown in Fig. S1. The qPCR analysis revealed the AIF1 transcription level was significantly higher in the CSFV Shimen-infected macrophages than in the control (mock infected) macrophages (Fig. 1B). IL6 transcription level was also assessed in CSFV Shimen-infected PAMs to identify the inflammatory responses triggered in response to infection, and a similar trend of increased transcriptional level was observed (Fig. 1C).

Detection of AIF1 and IL6 protein expression in macrophages following CSFV Shimen infection

Western blotting analysis revealed differences in AIF1 and IL6 protein expression levels between CSFV-infected and uninfected control cells, which demonstrated that AIF1 and IL6 levels increased significantly in the first 48 h after infection relative to the levels at the 0 time point (Figs. 2A–2H). Confocal microscopy was used to support the protein expression analysis. The colocalization of AIF1 and E2, an envelope glycoprotein of CSFV (Li et al., 2018), was observed (Figs. 2I–2P). The results of Western blotting quantification by ImageJ software supported that AIF1 and IL6 were significantly more abundant in the infected PAMs than in the uninfected control PAMs (Fig. S2). Meanwhile, the analysis by confocal laser scanning microscopy showed that there was significantly more AIF1 and IL6 in the CSFV Shimen-infected PAMs than in the uninfected control cells (Figs. 2Q–2X). A quantitative analysis was carried out to determine the significance of the confocal analysis (Fig. S3). These findings suggest that AIF1 plays a pro-inflammatory role in PAMs in response to infection by CSFV Shimen.

AIF1 stimulates PAMs 3D4/21 cells to augment IL6

We have shown that CSFV Shimen upregulated the AIF1 and IL6 expression in PAMs. A previous study showed AIF1 augments production of IL6 by a mouse macrophage line (Watano et al., 2001). To further elucidate the existence of certain association between AIF1 and IL6 levels on CSFV infection in PAMs, we transfected overexpressing AIF1 vectors into PAMs 3D4/21 cells to clarify cause–effect connection between those factors. First, overexpressing AIF1 plasmids (p-AIF1) were constructed (Fig. 3A) to compare its effects on AIF1 expression in PAMs 3D4/21 cells with the control groups (p-AIF1-NC or mock transfectants). Figure 3B showed that the expressing of AIF1 in PAMs 3D4/21 cells were significantly induced by overexpressing AIF1 plasmids (p-AIF1), and this effect was dose-dependent (Fig. 3C). Next, IL6 alterations of PAMs 3D4/21 cells by overexpressing AIF1 were examined, and relative transcription levels of IL6 analysis showed the significantly upregulated IL6 expression in PAMs 3D4/21 cells with the dose-dependent AIF1 expression (Fig. 3D). No significant results were obtained in the control groups (Fig. S4).

CSFV Shimen induces IL6 expression via AIF1

To further observe the impact of AIF1 on the inflammatory response in CSFV Shimen-infected PAMs 3D4/21 cells, after AIF1 expression in PAMs was silenced by siRNA, CSFV Shimen was inoculated in PAMs 3D4/21 cells comprised with the control groups to determine whether CSFV Shimen induces IL6 expression via AIF1. As shown is Fig. 4A, siRNA plasmids were constructed to compare the interference effects of three designed interference sequences (Fig. 4A). After the different plasmids were transfected (Fig. S5), PCR (Fig. S5) and qPCR analysis suggested that AIF1-p-Si1 has a significant inhibitory effect on AIF1 expression compared with the control AIF1-p-SiNC-transfected cells (Figs. 4B and 4C). Therefore, we transfected cells with AIF1-p-Si1 and AIF1-p-SiNC plasmids to investigate the role of AIF1 in CSFV Shimen-induced IL6 expression. The results showed that the levels of IL6 were blocked in AIF1-p-Si1-transfected PAMs 3D4/21 cells infected with CSFV Shimen compared to the levels in infected cells transfected with p-SiNC (Fig. 4D). In contrast, through the overexpression of recombinant AIF1 in PAMs, the levels of IL6 were up-regulated in p-AIF1-transfected PAMs 3D4/21 cells infected with CSFV Shimen compared to those transfected with p-AIF1-NC (Fig. 4E).

AIF1 confers susceptibility to CSFV Shimen in PAMs 3D4/21 cells

To investigate the relevance of AIF1 in the CSFV Shimen life cycle, the p-Si1 plasmid was used to target AIF1 in macrophages and compared to cells treated with a scrambled siRNA (AIF1-p-SiNC) and mock-treated cells before CSFV Shimen were inoculated into PAMs 3D4/21 cells. The qPCR analysis results showed accelerated replication of CSFV Shimen in the AIF1-p-SiNC-treated macrophages compared to a significant inhibition of CSFV Shimen growth in AIF1-p-Si1-treated macrophages, which was consistent with the results of the western blotting analysis (Fig. 5 and Fig. S6). Compare results were demonstrated in Fig. 5 that AIF1 over-expression is beneficial for the replication of CSFV Shimen. Together, these findings highlight a key role for cellular AIF1 in CSFV Shimen growth.