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.

Establishment and characterization of the immortalized porcine lung-derived mononuclear phagocyte cell line

“…ASFV is a highly pathogenic virus with a marked tropism for cells of the monocyte-macrophage lineage (21). Similar to the IPKM and IPIM cell lines reported in our recent studies (6,10), IPLuM were confirmed to be susceptible to ASFV infection and to facilitate the propagation of the virus very efficiently. It has been reported that the titers of replicating ASFV isolates were much lower in IPAM than in primary PAM (22).…”

“…At each passage, the cells were detached using TrypLE express solution (Thermo Fisher Scientific, Waltham, MA), and the number of harvested cells was measured using a Bio-Rad TC20 automated cell counter. Immortalized porcine intestinal macrophages (IPIM) and immortalized porcine kidney-derived macrophages (IPKM), which are highly sensitive to the field ASFV isolates and celladapted ASFV isolate reported in our recent studies (6,10), were also passaged in the same way.…”

Porcine Macrophage Markers and Populations: An Update

“…Similarly, Takenouchi et al established an immortalized cell line by transferring the SV40 large T antigen and porcine TERT genes into primary porcine kidney-derived macrophages using lentiviral vectors [ 287 ]. Using the same approach, these authors also successfully immortalized macrophages from lung and small intestine [ 288 , 289 ]. These cells retain many features of primary macrophages and are susceptible to infection by ASFV or PRRSV and may represent useful tools for studying the interaction of these porcine pathogens with macrophages [ 288 , 290 , 291 ].…”

“…Using the same approach, these authors also successfully immortalized macrophages from lung and small intestine [ 288 , 289 ]. These cells retain many features of primary macrophages and are susceptible to infection by ASFV or PRRSV and may represent useful tools for studying the interaction of these porcine pathogens with macrophages [ 288 , 290 , 291 ].…”

Isolation and propagation of bovine blood‐derived macrophages using a mixed culture with bovine endothelial B46 cells

“…To propagate blood‐derived macrophages (BMs) using such a mixed culture system, it is first necessary to identify appropriate feeder cells that can be used as an alternative to tissue‐derived stromal cells. Accumulating evidence suggests that α‐smooth muscle actin‐expressing myofibroblast‐like cells or vascular endothelial cells are capable of promoting the proliferation of BMs (Kitani et al, 2011; Takenouchi et al, 2022; Tanaka et al, 1993).…”