Expression profile of lncRNA in PAMs
To evaluate the performance of endogenous lncRNAs in PAMs, we designed a synthetic reference pool from three specific-pathogen-free (SPF) Landrace pigs by RNA-seq analysis. More specifically, our pool contained 2143 lncRNAs (350 known lncRNAs and 1793 novel lncRNAs) and 22566 mRNAs. To predict the function of these lncRNAs, we performed KEGG pathway analysis of cis- and trans-regulated predicted mRNAs. The result showed that enriched GO and KEGG pathways were mainly related to the inflammatory response, such as “MAPK signaling pathway”, “cytokine-cytokine receptor interaction”, “TNF signaling pathway”, “toll-like receptor signaling pathway”, and “Jak-STAT signaling pathway” (Fig. 1a). To make a connection between the enriched lncRNAs and mRNAs, we screened out the top 20 abundant mRNAs. As shown in Fig. 1b, majority of the first 20 abundant mRNAs were inflammatory response-related genes, such as Chemokine (C-X-C motif) ligand 8 (CXCL8), Ferritin light chain (FTL), S100A8, Chemokine (C-X-C motif) ligand 6 (CXCL6), Cathepsin S (CTSS), Galectin-3 (LGALS3), Elongation factor 1-alpha 1 (EEF1A), thioredoxin (trxA), Apolipoprotein E (APOE), C-type lysozyme enzyme (LYZ), Leukocyte surface antigen (CD53), Chemokine ligand x (CCLx), Superoxide dismutase 2 (SOD2), and Chemokine (C-X-C motif) ligand 2 (CXCL2). These data indicate that the PAMs are enriched in immune response-related lncRNAs and their predicted target genes, which allow them to respond quickly to invading organisms.
Validation the susceptibility of PAMs to PRRSV infection
To perform the functional analysis of porcine lncRNAs, we established in vitro model of inflammation by incubating PAMs with a high-pathogenic PRRSV strain HuN4. Cells were then collected for RNA sequence analysis. To confirm whether the PAMs were successfully infected by PRRSV, virus replication was determined by TCID50 and Western blot assay. The results showed that the virus titer obtained from PAMs supernatant was 5.25 log10 TCID50/ml (Fig. 2a) and the expression of N protein was also confirmed in PRRSV-treated PAMs (Fig. 2b; Additional file 1).
LncRNA expression profile in PRRSV-infected PAMs
Freshly isolated PAMs were treated with PRRSV HuN4 or mock, and followed by RNA-seq methodology. The results showed that the expression levels of 88 relatively abundant lncRNAs (FPKM > 1) were significantly altered upon virus incubation (Fig. 3a and Additional file 2). Among them, 36 lncRNAs were upregulated and 52 were downregulated (fold change>2.0, P-value<0.05). To further employ the differentially expressed (DE) lncRNAs upon PRRSV infection, the unsupervised hierarchical clustering analysis was used. Heat maps showed overt self-segregated clusters in PAMs treated with PRRSV and mock (Fig. 3b). To further verify the accuracy of RNA-seq results, we tested four upregulated lncRNAs (XLOC-022131, XLOC-022175, XLOC-019295, and XLOC-007149) and one downregulated lncRNA (XLOC-017089) by quantitative PCR (qPCR). As shown in Fig. 3c, XLOC-022131, XLOC-022175, XLOC-019295, and XLOC-007149 were significantly upregulated and XLOC-017089 was significantly downregulated, indicating the accuracy of RNA-seq data.
We next performed GO and KEGG pathway analyses to evaluate the function of DE lncRNAs. As shown in Fig. 3d, the upregulated lncRNAs were primarily associated with “NF−κB signaling pathway”, “toll−like receptor signaling pathway”, “MAPK signaling pathway”, “RIG−I−like receptor signaling pathway”, “Jak-STAT signaling pathway”, and “TNF signaling pathway”. The downregulated lncRNAs were enriched in “PI3K-Akt signaling pathway”, “chemokine signaling pathway”, and “MAPK signaling pathway” (Fig. 3e). The GO and KEGG pathway patterns suggest that DE lncRNAs are largely associated with the inflammation- and pathogen infection-induced immune responses upon PRRSV infection, indicating that these DE lnRNAs may play critical roles in regulating the virus-induced inflammatory responses in PAMs.
Gene expression signature in PRRSV-infected PAMs
Based on the analysis of RNA-seq data, we also generated volcano plots to visualize the DE profile of protein-coding mRNAs. Totally 407 mRNAs were differentially expressed (126 upregulated and 281 downregulated mRNAs) in PAMs after PRRSV treatment (Fig. 4a and Additional file 3). The hierarchical clustering analysis revealed that DE mRNAs were precisely distinguished by PRRSV-infected and mock-treated PAMs (Fig. 4b). Furthermore, we performed the GO and KEGG pathway analyses for DE mRNAs. Fig. 4c showed that the upregulated genes were enriched in “TNF signaling pathway”, “cytokine−cytokine receptor interaction”, “chemokine signaling pathway”, “rheumatoid arthritis”, “Toll−like receptor signaling pathway”, and “inflammatory bowel disease”. The most downregulated genes were enriched in pathways including “metabolic pathways” and “complement and coagulation cascades” (Fig. 4d). The pathway patterns determined by GO and KEGG analyses indicate that these DE genes are dominantly related to pathogen-induced inflammatory immune responses. Combined with lncRNA profiles, these results implicate that the DE lncRNAs may have indispensable regulatory functions in the DE genes in PAMs upon virus stimulation.
Identification of the regulatory function of lncRNAs in immune responses
Correlation network is an innovative tool to better integrate the co-expressed genes with the regulatory functions of lncRNAs. Here the correlation analysis encompassed 88 lncRNA nodes and 407 mRNA nodes with significant changes in the virus-stimulated PAMs. By using a Pearson Correlation, we identified numerous sets of DE genes with temporal co-expression patterns (Additional file 4). To annotate the function of DE lncRNAs, we selected four immune-related genes including CXCL2, IFI6, IFITM1, and CD163 as modules to unearth the corresponding regulatory lncRNAs.
CXCL2, as a powerful chemoattractant, is exclusively secreted by monocytes and macrophages, involving in neutrophil recruitment and other immune responses during inflammation and wound healing [24]. For the CXCL2-lncRNAs network, 17 lncRNAs positively correlated with CXCL2 expression were upregulated, while 24 lncRNAs negatively correlated with CXCL2 expression were downregulated in PRRSV-infected cells (Fig. 5a). The CXCL2 module indicates that significantly enriched lncRNAs may play a dual role in the macrophages for microbial killing and initiating tissue repair [25].
Numerous IFNs stimulated genes, transcriptionally induced as the downstream signaling molecule of IFNs, can inhibit the infection of multiple viral families. Among these antiviral genes, the IFN-inducible protein (IFI) family and IFN-induced transmembrane protein (IFITM) family have broad-spectrum antiviral functions. The IFITM family consists of IFITM1, 2, 3, 5, and 6 [26], which can block the replication and infection of enveloped viruses [27]. IFI6 can target viral replication at the endoplasmic reticulum or distinct membranous organelles [28]. Here, the IFI6 and IFITM1 were selected to build the lncRNA/mRNA co-expression network. For the IFN-inducible genes network, 27 lncRNAs positively correlated with IFITM1/IFI6 expression were upregulated, while 13 lncRNAs negatively correlated with IFITM1/IFI6 expression were downregulated in PRRSV-infected cells (Fig. 5b). Our results indicate that the DE lncRNAs associated with IFN-inducible genes have the potential to regulate the antiviral functions in macrophages.
CD163 has been identified as the essential receptor that mediates PRRSV entry [29, 30]. Therefore, the CD163-lncRNAs correlation network was analyzed. As shown in Fig. 5c, 16 lncRNAs negatively correlated with CD163 expression were upregulated, while 22 lncRNAs positively correlated with CD163 expression were downregulated in PRRSV infected cells. The CD163 module suggests that DE lncRNAs may play important roles in PRRSV entry by regulating the expression level of CD163.
Validation of differentially expressed lncRNAs by qPCR
To verify our correlation networks, qPCR was used to examine three significantly differentially expressed lncRNA-mRNA pairs in PRRSV-infected PAMs. As shown in Fig. 6, all three lncRNA-mRNA pairs, including lncRNA XLOC-022175 vs CXCL2 (co-upregulated), XLOC-019295 vs IFI6 (co-upregulated), and XLOC-017089 vs CD163 (co-downregulated) exhibited significant changes after virus infection, which coincided with the predicted correlation networks. Additionally, the relationship between these three lncRNAs and their neighboring genes was analyzed based on the genomic location and transcriptome expression profile (Fig. 6d; Table 2). Sequence analysis revealed that XLOC-22175 as a sense lncRNA located at the CXCL2 genomic locus, suggesting that the upregulated XLOC-022175 may promote the expression of CXCL2 (Fig. 6a). Sequence databases also showed that both XLOC-019295 and XLOC-017089 were antisense lncRNAs, and located near the genomic region of either IFI6 or CD163, indicating that both lncRNAs may facilitate the expression of IFI6 and CD163, respectively (Fig. 6b and c). Interestingly, XLOC-019295 was predicted to be a trans-regulatory element for the expression of distant gene IFITM1 (Table 2). Although the correlations of these three lncRNA-mRNA pairs were confirmed by qPCR, their potential immune regulatory function needs further investigation.