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. 2026 Jul 6;22:573. doi: 10.1186/s12917-026-05695-2

Ligation of the CD163 molecule inhibits the synthesis of type I and type III interferons in porcine alveolar macrophages

Liujun Zhang 1,2,3,4,✉, Man Tong 1, Aiyang Wang 1, Weizhen Chen 1, Bo Wang 1, Shaojun He 1,2,3,4, Hongjie Fan 1,2,3,4
PMCID: PMC13637252  PMID: 42410590

Abstract

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that poses a serious threat to the health of pigs worldwide. Porcine alveolar macrophages (PAMs) are the primary target cells for the replication of PRRSV. The CD163 molecule expressed on the surface of PAMs plays a crucial role in PRRSV infection by mediating viral particle uncoating and the release of genomic RNA. However, the role of CD163 in influencing the interferon (IFN) responses remains unidentified.

Results

Here, we found that in PAMs, the expression of IFN-α, IFN-β, and IFN-λ1 was downregulated by PRRSV infection, whereas the expression of interleukin-10 (IL-10) and CD163 was upregulated, suggesting that PRRSV inhibited type I and type III IFN responses. Ligation of CD163 in PAMs was found to downregulate the expression of IFN-α, IFN-β, and IFN-λ1 and upregulate the expression of IL-10, suggesting that CD163 inhibited the type I and type III IFN responses. Knockdown of CD163 in PAMs reversed the downregulation of IFN-α, IFN-β, and IFN-λ1 expression, as well as the upregulation of IL-10 expression caused by PRRSV infection.

Conclusions

In summary, our study showed that the CD163 molecule negatively regulated the IFN responses by downregulating the production levels of type I (IFN-α/β) and type III (IFN-λ1) IFNs in PAMs. It is speculated that the interaction between PRRSV and CD163 may contribute to the virus-triggered suppression of the IFN responses.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12917-026-05695-2.

Keywords: PRRSV, CD163, Interferons, Interleukin-10, PAMs

Background

Porcine reproductive and respiratory syndrome (PRRS) is extremely prevalent worldwide, causing significant economic losses to the pig farming industry [1]. PRRS is caused by the PRRS virus (PRRSV). The virus is a single-stranded positive-sense enveloped RNA virus belonging to the Arterivirus family [2]. There are two main genotypes of PRRSV: PRRSV-1 and PRRSV-2. Both genotypes can cause similar reproductive disorders and respiratory system problems in pigs, but their genomic RNA sequences share only approximately 60% [3]. PRRSV infection is mainly confined to a subset of porcine alveolar macrophages (PAMs). Furthermore, PRRSV can grow effectively in MARC-145 cells (a monkey kidney cell line), which are typically used for in vitro studies and for PRRSV proliferation [3, 4]. In fact, the entry of the PRRSV into host cells is a complex endocytosis and replication process mediated by cellular receptors. The presence or absence of cell-surface receptors determines the phenotype of cells infected with PRRSV. As of now, at least nine different molecules have been identified as potential receptors for the PRRSV, including sialoadhesin (Sn), vimentin, heat shock protein member 8, heparan sulfate, non-muscle myosin heavy chain 9, CD151, CD163, CD209, and T-cell immunoglobulin and mucin domain [5, 6]. These receptors play a crucial role in PRRSV infection of cells by mediating viral attachment, endocytosis, or uncoating. Among them, the CD163 molecule is responsible for uncoating the infectious PRRSV particles and releasing the genomic RNA. Deletion and blockade experiments targeting the scavenger receptor cysteine-rich (SRCR) domain indicated that SRCR5 in porcine CD163 is essential for PRRSV infection [7, 8].

Macrophages form the first line of defense in identifying and eliminating potential pathogens. These special cells possess the ability of phagocytosis, the capacity to degrade both their own and external substances, the ability to establish intercellular interactions, and the ability to produce inflammatory mediators [9]. Mature tissue macrophages express a variety of cell receptor proteins, including members of the SRCR superfamily. The SRCR domain has two variants, which divide the SRCR molecule into groups A and B. CD163 is a 130-kDa transmembrane glycoprotein belonging to the group B SRCR protein family. The molecule is mainly highly expressed in the cell subsets of the monocyte/macrophage lineage [9, 10]. CD163 has been characterized as a receptor for the hemoglobin-haptoglobin (Hb-Hp) complex. It can mediate the endocytosis of this Hb-Hp complex and intercellular interactions between macrophages and developing erythroblasts [11]. CD163 has also been proven to play a significant role in the infection process of various pathogens, including viruses and bacteria. Some findings have identified CD163 as a putative macrophage receptor for PRRSV, African swine fever virus, and gram-positive/gram-negative bacteria [12–14]. In addition, an increasing body of evidence indicates that CD163 participates in the inflammatory response by regulating the expression levels of inflammatory factors, such as nitric oxide (NO), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, and IL-10 [15, 16]. However, little is known about CD163 in the regulation of the interferon (IFN) responses.

Type I (IFN-α/β) and type III (IFN-λs) IFNs exhibit strong anti-PRRSV activity [17]. Simultaneously, porcine Sn inhibits type I IFN production, thereby supporting PRRSV infection [18]. Porcine CD163 has also been successfully cloned and expressed. It is composed of a signal peptide, nine consecutive SRCR sequences (numbered 1 to 9), a transmembrane domain, and an intracellular cytoplasmic tail [19]. This study aims to explore the influence of porcine CD163 on the type I and type III IFN responses.

Results

PRRSV reduces the expression levels of type I and type III IFNs in PAMs

As shown in Fig. 1a-f, at 12 to 48 h post-infection, the mRNA and protein levels of IFN-α, IFN-β, and IFN-λ1 in the virus-infected group were significantly lower than those in the uninfected mock group. Conversely, the mRNA and protein levels of IL-10 in the virus-infected group were substantially higher than those in the uninfected mock group (Fig. 1g and h). The replication kinetics of the virus in PAMs showed that the virus production gradually increased over time from 12 to 48 h after infection, and reached its peak at 48 h post-infection (Fig. 2). In addition, Fig. 3 showed that at 12 to 48 h after stimulation, the mRNA and protein levels of IFN-α, IFN-β, and IFN-λ1 in Poly (I: C)-stimulated group with virus infection were significantly lower than those in Poly (I: C)-stimulated group and the mRNA and protein levels of IL-10 in Poly (I: C)-stimulated group with virus infection were significantly higher than those in Poly (I: C)-stimulated group. These data suggested that PRRSV infection reduced the transcripts and secretion of type I and type III IFNs in PAMs.

Fig. 1.

Fig. 1

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The influence of PRRSV infection or CD163 ligation on cytokine production in PAMs. The cells were treated with PRRSV, Poly (I: C), rabbit negative IgG, or anti-CD163 SRCR5 IgG. At the specified time point, cells and the supernatants were harvested. The mRNA transcription and protein secretion of cytokines were detected by relative quantitative RT-PCR and ELISA, respectively. a IFN-α mRNA; b IFN-α protein; c IFN-β mRNA; d IFN-β protein; e IFN-λ1 mRNA; f IFN-λ1 protein; g IL-10 mRNA; h IL-10 protein. Each group has three replicates (N = 3), and results are presented as means ± SD from three independent experiments

Fig. 2.

Fig. 2

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The proliferation kinetics curve of PRRSV in PAMs. The cells were infected with PRRSV. At the specified time point, infected cells and the supernatants were harvested. Viral replication in cells and cell supernatants was detected by real-time RT-PCR, virus titer determination, and western blot. a The RNA copies of PRRSV within the cells; b The TCID50 titers of PRRSV in the cell supernatants; c The level of N protein of PRRSV within the cells; d The results of the densitometric analysis in Fig. 2c. Each group has three replicates (N = 3) and the results are presented as the means ± SD of three independent experiments

Fig. 3.

Fig. 3

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The influence of PRRSV infection or CD163 ligation on cytokine production induced by Poly (I: C) in PAMs. The cells were incubated with PRRSV, rabbit negative IgG, or anti-CD163 SRCR5 IgG for 1 h, then treated with Poly (I: C) for 12 to 48 h. At the specified time point, cells and the supernatants were harvested. The mRNA transcription and protein secretion of cytokines were detected by relative quantitative RT-PCR and ELISA, respectively. a IFN-α mRNA; b IFN-α protein; c IFN-β mRNA; d IFN-β protein; e IFN-λ1 mRNA; f IFN-λ1 protein; g IL-10 mRNA; h IL-10 protein. Each group has three replicates (N = 3), and results are presented as means ± SD from three independent experiments

PRRSV increases the expression level of the CD163 molecule in PAMs

As shown in Fig. 4, at 12 to 48 h after infection, the mRNA and protein levels of CD163 in the virus-infected group were significantly higher than those in the mock-uninfected group, suggesting that PRRSV infection increased CD163 expression in PAMs.

Fig. 4.

Fig. 4

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The influence of PRRSV infection on the expression level of the CD163 molecule in PAMs. The cells were infected with PRRSV. At the specified time point, the cells were harvested. The transcription and protein levels of CD163 were detected by relative quantitative RT-PCR and western blot, respectively. a CD163 mRNA; b CD163 protein; c The results of the densitometric analysis in Fig. 4b. Each group has three replicates (N = 3) and the results are presented as the means ± SD of three independent experiments

Ligation of the CD163 molecule reduces the expression levels of type I and type III IFNs in PAMs

As shown in Fig. 1, at 12 to 48 h post-treatment, the mRNA and protein levels of IFN-α, IFN-β, and IFN-λ1 in the anti-CD163 SRCR5 IgG-treated group were significantly lower than those in the rabbit negative IgG-treated group. In contrast, the mRNA and protein levels of IL-10 in the anti-CD163 SRCR5 IgG-treated group were substantially higher than those in the rabbit negative IgG-treated group. Additionally, Fig. 3 showed that at 12 to 48 h after stimulation, the mRNA and protein levels of IFN-α, IFN-β, and IFN-λ1 in Poly (I: C)-stimulated group with anti-CD163 SRCR5 IgG treatment were significantly lower than those in Poly (I: C)-stimulated group with rabbit negative IgG treatment and the mRNA and protein levels of IL-10 in Poly (I: C)-stimulated group with anti-CD163 SRCR5 IgG treatment were significantly higher than those in Poly (I: C)-stimulated group with rabbit negative IgG treatment. These data suggested that ligation of CD163 reduced the transcripts and secretion of type I and type III IFNs in PAMs.

Knockdown of the CD163 molecule reverses the reduction of expression levels of type I and type III IFNs caused by PRRSV in PAMs

As shown in Fig. 5a, the mRNA level of CD163 in the CD163 siRNA-transfected group was significantly lower than that in the negative control siRNA-transfected group at 24 to 60 h after transfection. The protein level of CD163 in the CD163 siRNA-transfected group was significantly lower than that in the negative control siRNA-transfected group at 36 h after transfection (Fig. 5b and c). These data suggested that CD163 siRNA effectively reduced CD163 expression in PAMs. After 36 h of transfection, the cells were infected with PRRSV. Figure 6a-f showed that at 12 to 24 h after infection, the mRNA and protein levels of IFN-α, IFN-β, and IFN-λ1 in the virus-infected group with CD163 siRNA transfection were significantly higher than those in the virus-infected group with negative control siRNA transfection. The mRNA and protein levels of IL-10 in the virus-infected group with CD163 siRNA transfection were significantly lower than those in the virus-infected group with negative control siRNA transfection (Fig. 6g and h). These data suggested that knockdown of CD163 reversed the reduction of transcripts and secretion of type I and type III IFNs caused by PRRSV infection in PAMs.

Fig. 5.

Fig. 5

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The knockdown of the CD163 molecule in PAMs. The cells were transfected with either CD163 siRNA or negative control siRNA. At the specified time point, the cells were harvested. The transcription and protein levels of CD163 were detected by relative quantitative RT-PCR and western blot, respectively. a CD163 mRNA; b CD163 protein; c The results of the densitometric analysis in Fig. 5b. Each group has three replicates (N = 3) and the results are presented as the means ± SD of three independent experiments

Fig. 6.

Fig. 6

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The influence of knockdown of the CD163 molecule on cytokine production induced by PRRSV in PAMs. The cells were transfected with either CD163 siRNA or negative control siRNA. After 36 h, transfected cells were infected with PRRSV. At the specified time point, cells and the supernatants were harvested. The mRNA transcription and protein secretion of cytokines were detected by relative quantitative RT-PCR and ELISA, respectively. a IFN-α mRNA; b IFN-α protein; c IFN-β mRNA; d IFN-β protein; e IFN-λ1 mRNA; f IFN-λ1 protein; g IL-10 mRNA; h IL-10 protein. Each group has three replicates (N = 3), and results are presented as means ± SD from three independent experiments

Knockdown of the CD163 molecule decreases the production levels of PRRSV in PAMs

As shown in Fig. 7, the RNA copy and N protein levels of PRRSV in the virus-infected group with CD163 siRNA transfection were significantly lower than those in the virus-infected group with negative control siRNA transfection. These data suggested that CD163 knockdown decreased PRRSV production in PAMs.

Fig. 7.

Fig. 7

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The influence of knockdown of the CD163 molecule on the proliferation of PRRSV in PAMs. The cells were transfected with either CD163 siRNA or negative control siRNA. After 36 h, transfected cells were infected with PRRSV. At the specified time point, cells and the supernatants were harvested. Viral replication in cells and cell supernatants was detected by real-time RT-PCR, virus titer determination, and western blot. a The RNA copies of PRRSV within the cells; b The TCID50 titers of PRRSV in the cell supernatants; c The level of N protein of PRRSV within the cells; d The results of the densitometric analysis in Fig. 7c. Each group has three replicates (N = 3) and the results are presented as the means ± SD of three independent experiments

Discussion

Interferons (IFNs) are a set of pleiotropic innate immune cytokines that play a crucial role in rapidly responding to pathogens, regulating the immune system, and providing protection against cancer, among other functions. They are ubiquitously produced by a wide variety of cells in response to viral infection, damage, or cellular stress [20]. IFNs are divided into three distinct subtypes, including type I (IFN-α/β), type II (IFN-γ), and type III (IFN-λs). Type I and type III IFNs are major components of antiviral immunity, inducing similar downstream signaling cascades [21]. IFNs effectively limit PRRSV replication in host cells. Therefore, the virus employs multiple mechanisms to counteract IFN responses for enhancing its survival [22, 23]. Early reports showed that PAMs infected with PRRSV exhibited delayed, low-level type I IFN activity. The level of IFN-α in infected pigs was relatively low and undetectable in lung tissues where PRRSV actively replicated [22, 24, 25]. PRRSV is also reported to inhibit the secretion of IFN-α by porcine plasmacytoid dendritic cells and the transcript of IFN-λ1 in PAMs [26, 27]. In this study, we observed downregulation of IFN-α, IFN-β, and IFN-λ1 production in PAMs following infection with the PRRSV-2 AP-4 strain. Moreover, the AP-4 strain reduced Poly (I: C)-induced IFN-α, IFN-β, and IFN-λ1 secretion in PAMs. These findings further supported the notion that PRRSV inhibited IFN responses. IL-10 is a crucial anti-inflammatory cytokine produced by various cell types. It plays a critical role in regulating immune responses. IL-10 has been implicated in immunosuppression by suppressing the expression of pro-inflammatory cytokines and countering the adaptive immune response [28, 29]. Here, we found that infection with the AP-4 strain increased IL-10 production in PAMs, consistent with previous findings [27, 30].

The CD163 molecule has been described as a functional receptor essential for PRRSV replication in host cells by facilitating viral entry and uncoating [5, 6, 14]. The SRCR5 domain of CD163 has been confirmed as a key structural domain involved in interaction with PRRSV particles [7, 8, 31]. After infection with a virulent PRRSV-1 strain, CD163 expression in live PAMs has been reported to be downregulated [32]. However, the modulation of CD163 expression by PRRSV may be strain-dependent. Jiang et al. reported that the mRNA level of the CD163 gene was significantly higher in the lung tissues of PRRSV-2 strain-infected Chinese Dapulian pigs than in uninfected pigs [33]. We found that the PRRSV-2 AP-4 strain increased CD163 receptor expression in PAMs in vitro. Multiple studies have shown that IL-10 can strongly promote CD163 expression in monocytes and macrophages [34–37]. Hence, the upregulation of CD163 level in the AP-4 strain-infected PAMs might be due to virus-induced IL-10 secretion. Since CD163 expression modulation by PRRSV is strain-dependent, testing multiple PRRSV strains will help in strengthening the conclusion. Our future studies will further validate these observations with both PRRSV-1 and PRRSV-2 isolates. Previous research confirmed that cross-linking of the CD163 receptor with specific monoclonal antibodies for CD163 triggered the production of several anti-inflammatory and pro-inflammatory mediators, including IL-10, IL-1β, IL-6, NO, and TNF-α, indicating a direct role for CD163 in the immune response and/or host defense [12, 15, 16]. Furthermore, antibodies against CD163 inhibited PRRSV infection and the related nuclear factor (NF)-κB pathway in PAMs and MARC-145 cells, both before and after virus attachment, suggesting a potential effect of CD163 on the IFN responses [38]. Since NF-κB is essential for IFN transcription, CD163 may inhibit IFN responses by negatively regulating the NF-κB or IFN regulatory factor 3/7 pathways. Additionally, CD163’s cytoplasmic tail may recruit phosphatases such as Src homology 2 domain-containing protein tyrosine phosphatase-1 or suppressor of cytokine signaling proteins to suppress IFN production [38, 39].

In the present study, we treated PAMs with anti-CD163 SRCR5 antibodies to activate the CD163 receptor, and then analyzed the expression of type I and type III IFNs. We found that the production levels of IFN-α, IFN-β, and IFN-λ1 in the CD163-activated PAMs were significantly lower than those in inactivated cells. Meanwhile, the selective activation of CD163 in PAMs reduced the release of IFN-α, IFN-β, and IFN-λ1 induced by Poly (I: C). These data indicated that the CD163 molecule inhibited IFN responses. In addition, we observed that CD163 increased IL-10 secretion in PAMs. IL-10 is a known inhibitor of IFN production [40]. Our current data cannot fully distinguish CD163’s direct and IL-10’s indirect effects on IFN responses. Consequently, it will be interesting to further determine whether CD163 directly inhibits IFNs or acts indirectly by IL-10 induction. Further, we also found that CD163 knockdown in PAMs reversed the downregulation of IFN-α, IFN-β, and IFN-λ1 expression induced by PRRSV and reduced PRRSV proliferation. These data suggested that CD163 might be involved in the inhibition process of type I and type III IFN production triggered by the PRRSV. In this study, antibody-mediated cross-linking of CD163 in the absence of any viral component significantly downregulated IFN-α/β/λ1 production and upregulated IL-10 secretion in PAMs. This demonstrated that CD163 engagement alone might be sufficient to trigger intracellular signals that negatively regulated IFN expression, independent of viral replication or entry. However, CD163 is also a well-established entry/uncoating receptor for PRRSV [5–8]. Thus, in CD163 knockdown experiments, the restored IFN production could be explained by either: (i) Loss of CD163 signaling that normally suppresses IFN expression (direct effect), or (ii) Reduced viral entry/replication, leading to decreased viral antagonism of IFN responses (indirect effect). Distinguishing between direct CD163-mediated signaling and indirect effects due to reduced viral burden will be of great significance.

In conclusion, our studies showed that PRRSV inhibited the expression of type I and type III IFNs and induced CD163 expression in PAMs during infection. Ligation of CD163 in PAMs also inhibited the expression of type I and type III IFNs. Knockdown of CD163 reversed the virus-triggered decrease in type I and type III IFN production. Here, we assumed that the interaction of CD163 with PRRSV activated the CD163 signaling pathway in PAMs. Therefore, it was possible that PRRSV-CD163 interaction was responsible for the virus-triggered inhibition of the IFN responses. Although our data demonstrate an association between CD163 receptor signaling and reduced type I/III IFN production, the precise molecular pathway by which CD163 engagement leads to IFN suppression remains to be fully elucidated. Moreover, our conclusions on IFN expression suppression by CD163 implied downstream effects on IFN-stimulated genes (ISGs), but direct measurement was lacking. Future studies will quantitatively assess ISG expression levels in CD163-activated or CD163-knockdown PAMs to more broadly confirm the functional, negatively antiviral outcomes of the CD163 molecule.

Conclusions

PRRSV decreased the production of IFN-α, IFN-β, and IFN-λ1, while increasing IL-10 and CD163 levels in PAMs. Likewise, CD163 ligation reduced IFN-α, IFN-β, and IFN-λ1 production and raised IL-10 levels in PAMs. In contrast, CD163 knockdown in PAMs reversed the PRRSV-induced downregulation of IFN-α, IFN-β, and IFN-λ1, as well as the upregulation of IL-10. Together, these findings suggested that CD163 negatively regulated innate antiviral responses by inhibiting the synthesis of type I and type III IFNs in PAMs. Therefore, the PRRSV-CD163 interaction may contribute to viral suppression of IFN antiviral responses.

Methods

Cells and virus strain

The nine PRRSV-negative five-week-old pigs used in this study were sourced from commercial pig farms in Fengyang County, Anhui Province, China. PAMs were obtained from these PRRSV-negative piglets via bronchoalveolar lavage. The cells were re-suspended in RPMI-1640 medium (HyClone, USA) containing 10% fetal bovine serum (FBS) (HyClone), 100 µg/mL streptomycin, and 100 U/mL penicillin for later use. PRRSV-2 AP-4 strain (GenBank No.: PX715237) was isolated from the spleen of the infected pig. The virus titers were quantified as 50% tissue culture infectious dose (TCID50) in MARC-145 cells [31].

Antibodies

Anti-CD163 SRCR5 polyclonal antibody (pAb) with an ELISA titer of 1:12800 was derived from New Zealand White Rabbits inoculated with recombinant SRCR5 domain protein of the pig CD163 molecule. Anti-CD163 SRCR5 IgG was purified from rabbit anti-CD163 SRCR5-specific pAb. Rabbit negative IgG was extracted from the serum of healthy rabbits. The rabbit IgG antibody was treated with an endotoxin removal agent before use and confirmed to be endotoxin-free using an endotoxin detection kit (Sangon Biotech, China). Anti-β-actin antibody, anti-PRRSV N protein antibody, and anti-rabbit IgG antibody (HRP-labeled) were from Absin (China), Bioss (China), and Earthox (USA), respectively.

Reagents

Poly (I: C) and Lipofectamine 3000 transfection reagent were provided by InvivoGen (France) and Invitrogen (USA), respectively. TRIzol reagent and reverse transcription (RT) kits were purchased from TaKaRa (Japan).

PRRSV infection of PAMs and ligation of the CD163 molecule in PAMs

PAMs (150000 cells) cultured for 12 h at 37 °C in 24-well plates (NEST, China) were treated with PRRSV of 0.01 multiplicity of infection (MOI), Poly (I: C) (100 µg/mL), rabbit negative IgG (300 µg/mL), or anti-CD163 SRCR5 IgG (300 µg/mL) for 12 to 48 h. Untreated mock PAMs were used as the control group. Cells and supernatants were collected at the indicated time points (12–48 h) and used for subsequent detection of cytokine expression and determination of PRRSV production.

PRRSV infection of poly (I: C)-stimulated PAMs and ligation of the CD163 molecule in poly (I: C)-stimulated PAMs

PAMs (150000 cells) cultivated for 12 h at 37 °C in 24-well plates (NEST) were incubated with PRRSV (0.01 MOI), rabbit negative IgG (300 µg/mL), or anti-CD163 SRCR5 IgG (300 µg/mL) for 1 h and then stimulated by Poly (I: C) (100 µg/mL) for 12 to 48 h. Unstimulated mock PAMs and Poly (I: C)-stimulated PAM cells served as the control groups. Cells and supernatants were collected and used for subsequent detection of cytokine expression and determination of PRRSV production.

PRRSV infection of CD163-silenced PAMs

PAMs (150000 cells) were kept for 12 h at 37 °C in 24-well plates (NEST) and transfected with either CD163 small interfering RNA (siRNA) or a negative control siRNA at 40 pmol using Lipofectamine 3000. After 24–60 h, CD163 expression in transfected cells was detected by relative quantitative RT-PCR and western blot. Then, after 36 h, transfected cells were infected with 0.01 MOI of PRRSV for 12 to 24 h. Cells and supernatants were collected and used for subsequent detection of cytokine expression and determination of PRRSV production.

RNA extraction, relative quantitative RT-PCR, and real-time PCR

Total cell RNA was extracted using TRIzol, and the cDNA was synthesized using RT kits. Relative quantitative RT-PCR for mRNA transcripts of IFN-α, IFN-β, IFN-λ1, IL-10, and CD163 was performed in the Applied Biosystems Q5 Real-Time System (USA). The reaction conditions were as follows: 2 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 20 s at 60 °C. Simultaneously, real-time PCR was used to detect PRRSV viral RNA, as described by Zhang et al. [41]. The 2−∆∆CT method was used for analyzing mRNA levels of target genes.

ELISA for measuring the production of cytokines in culture cell supernatants

The commercial ELISA kits for IFN-α (MyBioSource, USA), IFN-β (Invitrogen, USA), IFN-λ1 (MyBioSource, USA), and IL-10 (Invitrogen, USA) were used to quantify the production levels of the corresponding immune cytokines in cell supernatants, referring to the manufacturer’s instructions.

Western blot for the detection and analysis of the target proteins

PAM cell lysates were isolated by 10% SDS-PAGE. The separated proteins on the gel were transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk. The blocked membranes were incubated with the primary rabbit-derived antibodies: anti-CD163 SRCR5 IgG (3 µg/mL), anti-PRRSV N protein antibody (1:2000 dilution), or anti-β-actin antibody (1:3000 dilution) overnight at 4 °C, and the secondary antibody: anti-rabbit IgG antibody (1:10000 dilution) at 37 °C for 1 h. The target proteins on the membranes were visualized using ECL reagents of GE Healthcare (UK). β-actin protein was used as a standard to normalize the target proteins.

Statistical analysis

The data are shown as the means ± standard deviations (SD) from at least three independent experiments. A two-way ANOVA with Bonferroni post hoc tests was performed in GraphPad Prism 5.0 for pairwise multiple comparisons. Results were considered statistically significant when the P value was less than 0.05 (*** P < 0.001, ** P < 0.01, * P < 0.05).

Supplementary Information

Supplementary Material 1. (85.9KB, pdf)
Supplementary Material 2. (98.6KB, pdf)
Supplementary Material 3. (78.6KB, pdf)
Supplementary Material 4. (90.5KB, pdf)

Acknowledgements

Not applicable.

Abbreviations

PRRSV

Porcine reproductive and respiratory syndrome virus

PAMs

Porcine alveolar macrophages

IFN

Interferon

IL-10

Interleukin-10

SRCR

Scavenger receptor cysteine-rich

Hb-Hp

Hemoglobin-haptoglobin

NO

Nitric oxide

TNF-α

Tumor necrosis factor-α

FBS

Fetal bovine serum

TCID50

50% tissue culture infectious dose

siRNA

Small interfering RNA

Authors' contributions

LZ and MT served as the primary investigators for this study. AW, WC, and BW conducted the data analysis. SH provided valuable suggestions and resources. LZ and HF supervised the experiments and secured funding. LZ was responsible for the writing, review, and editing of the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (32202817), the Anhui Province Key Project for Excellent Young Teacher Training in Higher Education Institution (YQZD2024033), the 2024 Year Youth Top Talent Project of Anhui Provincial Higher Education Institution, the Talent Introduction Project of Anhui Science and Technology University (DKYJ202103), the Excellent Research Innovation Team in Universities in Anhui Province (2022AH010088), and the Veterinary Science Peak Discipline Project of Anhui Science and Technology University (XK-XJGF002).

Data availability

All the data are included in the published papers.

Declarations

Ethics approval and consent to participate

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Anhui Science and Technology University, China (License Number: 20220102), and performed in accordance with established guidelines and regulations for scientific purposes. Written informed consent was obtained from the owners for animal participation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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