Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections

Abstract

Macrophages are a heterogeneous population of cells that are present in all vertebrate tissues. They play a key role in the innate immune system, and thus, in vitro cultures of macrophages provide a valuable model for exploring their tissue-specific functions and interactions with pathogens. Porcine macrophage cultures are often used for the identification and characterization of porcine viral pathogens. Recently, we have developed a simple and efficient method for isolating primary macrophages from the kidneys and livers of swine. Here, we applied this protocol to fetal porcine intestinal tissues and demonstrated that porcine intestinal macrophages (PIM) can be isolated from mixed primary cultures of porcine small intestine-derived cells. Since the proliferative capacity of primary PIM is limited, we attempted to immortalize them by transferring the SV40 large T antigen and porcine telomerase reverse transcriptase genes using lentiviral vectors. Consequently, immortalized PIM (IPIM) were successfully generated and confirmed to retain various features of primary PIM. We further revealed that IPIM are susceptible to infection by the African swine fever virus and the porcine reproductive and respiratory syndrome virus and support their replication. These findings suggest that the IPIM cell line is a useful tool for developing in vitro models that mimic the intestinal mucosal microenvironments of swine, and for studying the interactions between porcine pathogens and host immune cells.

Keywords: African swine fever virus; immortalized porcine intestinal macrophages; in vitro model; porcine reproductive and respiratory syndrome virus; porcine small intestine macrophages.

Figure 1

Immunocytochemical characterization of a mixed primary culture of fetal porcine small intestine-derived cells and isolated PIM. Primary intestinal cells cultured in T-75 tissue culture flasks (A) or TC-dishes (B) were observed under a phase-contrast microscope. The cells were seeded in 8-well-chamber slides, fixed using 4% paraformaldehyde phosphate buffer solution, and immunostained with specific antibodies against mesenchymal (αSMA and vimentin), macrophage (CD172a), or epithelial (CK18 and CK19) cell markers [(C), brown]. The PIM isolated from the supernatant samples of the mixed primary culture were immunostained with specific antibodies against macrophage (Iba1, CD204, and CD172a) or epithelial (CK18 and CK19) cell markers [(D), brown)]. Multinucleated giant cells were sometimes observed in the PIM culture [(D), white asterisks]. All nuclei were counterstained with hematoxylin [(C,D), blue]. Images are representative of two independent experiments.

Figure 2

Establishment of IPIM and comparison of their features with IPKM. The morphologies of IPIM and IPKM were observed under a phase-contrast microscope (A). The cumulative population doublings of the IPIM were plotted against the duration of the culture period (in days) (B). PCR-based sex identification based on the porcine amelogenin (AMEL) genes was performed using genomic DNA from the IPIM and IPKM, and PCR products derived from AMELX (520 bp) and AMELY (350 bp) were analyzed by agarose gel electrophoresis (C).

Figure 3

Immunocytochemical characterization of IPIM. The IPIM were seeded in 8-well-chamber slides and cultured for 1 day (A) or 3 days (B). Then, the cells were fixed using 4% paraformaldehyde phosphate buffer solution, and immunostained with specific antibodies against cell markers of macrophages (Iba1, CD172a, CD204, CD203a, and CD16) or specific subpopulations of macrophages (CD163, CD169, and MHC-II) (brown). No specific staining was observed if the cells were treated without primary antibodies [NC: negative control in (A,B)]. All nuclei were counterstained with hematoxylin (blue). Images are representative of three independent experiments.

Figure 4

Production of IL-1α and IL-1β in MDP- or LPS-stimulated IPIM. Dose-dependent production of pro-IL-1α and pro-IL-1β was detected in cell lysates from IPIM that had been stimulated with MDP or LPS for 3 days (second and fourth panels). The secretion of their mature forms (mIL-1α and mIL-1β) into the culture supernatant was detected in the LPS-treated IPIM (first and third panels). Actin was detected as an internal control for protein loading in each lane (fifth panel). Data are representative of three independent experiments.

Figure 5

Cytopathic effects and hemadsorption assays of ASFV-inoculated IPIM. Cells were mock-inoculated (A,C) or inoculated with the Armenia07 isolate (MOI = 0.1) (B,D) in the absence (A,B) or presence (C,D) of porcine red blood cells. Cytopathic effects (B) and rosette formation [(D), white arrows] were detected in Armenia07-infected cells at 2 dpi. Images are representative of at least three independent experiments.

Figure 6

Comparison of the ASFV production in IPIM and PAM. Cell cultures were infected with Armina07 (upper), Kenya05/Tk-1 (middle), and Lisbon60V isolates (lower) (MOI = 0.01). The culture supernatant samples were collected at the indicated timepoints. Viral production in the IPIM (closed circle) and PAM (open triangle) cell cultures were estimated by titration experiments with the IPIM. Data represent the mean and standard deviations of three experiments. Asterisks indicate statistically significant differences in the viral production between the IPIM and PAM cell cultures (*p < 0.05).

Figure 7

Detection of PRRSV antigens using an immunofluorescence assay, cytopathic effects, and viral growth curve for PRRSV-inoculated IPIM. A direct immunofluorescence assay was performed with FITC-conjugated anti-PRRSV monoclonal antibodies using mock-inoculated (A) and PRRSV-inoculated (B) cells at 3 dpi. The cytopathic effects of the PRRSV were also evaluated in mock-inoculated (C) and PRRSV-inoculated (D) cells at 7 dpi under a phase-contrast microscope. After inoculation with PRRSV, culture supernatant samples were collected at the indicated timepoints and used for titration experiments with the IPIM (E). Data are expressed as the mean ± SEM of three experiments (E).