Abstract:Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons o… Show more
“…Animal procedures and the isolation of alveolar macrophages and peripheral blood mononuclear cells (PBMCs) were conducted as described previously (29,(31)(32)(33)(34)(35)(36)(37). PBMCs were isolated using a 60% Ficoll-Paque Plus gradient (GE Healthcare, Piscataway, NJ).…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…Mediators and conditions for polarization of porcine monocytic cells were applied as previously described (1)(2)(3)(4)(5)29). In brief, Ms and DCs were stimulated with the mediators LPS, IFN-␥, IL-4, IL-10, IFN-␣, and IFN- at 20 ng/ml, and blood monocytes (BMs) were stimulated with either GM-CSF at 50 ng/ml or M-CSF at 100 ng/ml for 30 h. All mediators (purchased from R&D Systems, Minneapolis, MN, or Sigma-Aldrich, St. Louis, MO) were dissolved in 1ϫ Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen) containing 1% bovine serum albumin (BSA) (fraction V, cold ethanol precipitated; Sigma) and applied (1:100) to the cultured cells.…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…The expression of marker genes relative to each activation status and antiviral state was revealed using next-generation transcriptome sequencing (RNA-Seq) for genome-wide screening and confirmed family-wide using real-time reverse transcription-PCR (RT-PCR) assays. For RNA-Seq, equal quantities of primary alveolar macrophages were polarized for 30 h individually by the verified procedure described above and further infected for 5 h with a PRRSV strain as previously described (29). RNA preparation, RNA-Seq performance, and differentially expressed gene (DEG) analysis were done as previously described (29).…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…For RNA-Seq, equal quantities of primary alveolar macrophages were polarized for 30 h individually by the verified procedure described above and further infected for 5 h with a PRRSV strain as previously described (29). RNA preparation, RNA-Seq performance, and differentially expressed gene (DEG) analysis were done as previously described (29). In brief, 3 ϫ 10 7 cells at each activation status (including the control DPBS mock stimulation) were pooled from three technical replicates representing cells obtained from four outbred pigs.…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…Few studies have focused on the activation status of porcine monocytic cells or how cell activation status modulates antiviral immunity (27,28). Notably, in this study we focused on examining Gene Ontology (GO) analysis based on comparative transcriptomes revealed in macrophages at different activation statuses upon viral infection, rather than a transcriptomic comparison between infected and noninfected tissues/cells, which has been well documented in previous studies (29,30). In this context, we used a multiplex cytokine assay and transcriptomic sequencing (RNA-Seq) analysis to profile gene response pathways in porcine monocytic cells polarized to typical activation statuses and antiviral states.…”
IMPORTANCE
Activation statuses of monocytic cells, including monocytes, macrophages (Ms), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic, including our focus, PRRSV, which alone causes nearly $800 million economic loss annually in the U.S. swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine the intricate interaction of viral infection with activation statuses and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.
“…Animal procedures and the isolation of alveolar macrophages and peripheral blood mononuclear cells (PBMCs) were conducted as described previously (29,(31)(32)(33)(34)(35)(36)(37). PBMCs were isolated using a 60% Ficoll-Paque Plus gradient (GE Healthcare, Piscataway, NJ).…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…Mediators and conditions for polarization of porcine monocytic cells were applied as previously described (1)(2)(3)(4)(5)29). In brief, Ms and DCs were stimulated with the mediators LPS, IFN-␥, IL-4, IL-10, IFN-␣, and IFN- at 20 ng/ml, and blood monocytes (BMs) were stimulated with either GM-CSF at 50 ng/ml or M-CSF at 100 ng/ml for 30 h. All mediators (purchased from R&D Systems, Minneapolis, MN, or Sigma-Aldrich, St. Louis, MO) were dissolved in 1ϫ Dulbecco's phosphate-buffered saline (DPBS) (Invitrogen) containing 1% bovine serum albumin (BSA) (fraction V, cold ethanol precipitated; Sigma) and applied (1:100) to the cultured cells.…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…The expression of marker genes relative to each activation status and antiviral state was revealed using next-generation transcriptome sequencing (RNA-Seq) for genome-wide screening and confirmed family-wide using real-time reverse transcription-PCR (RT-PCR) assays. For RNA-Seq, equal quantities of primary alveolar macrophages were polarized for 30 h individually by the verified procedure described above and further infected for 5 h with a PRRSV strain as previously described (29). RNA preparation, RNA-Seq performance, and differentially expressed gene (DEG) analysis were done as previously described (29).…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…For RNA-Seq, equal quantities of primary alveolar macrophages were polarized for 30 h individually by the verified procedure described above and further infected for 5 h with a PRRSV strain as previously described (29). RNA preparation, RNA-Seq performance, and differentially expressed gene (DEG) analysis were done as previously described (29). In brief, 3 ϫ 10 7 cells at each activation status (including the control DPBS mock stimulation) were pooled from three technical replicates representing cells obtained from four outbred pigs.…”
Section: Animals and Isolation Of Primary Cellsmentioning
confidence: 99%
“…Few studies have focused on the activation status of porcine monocytic cells or how cell activation status modulates antiviral immunity (27,28). Notably, in this study we focused on examining Gene Ontology (GO) analysis based on comparative transcriptomes revealed in macrophages at different activation statuses upon viral infection, rather than a transcriptomic comparison between infected and noninfected tissues/cells, which has been well documented in previous studies (29,30). In this context, we used a multiplex cytokine assay and transcriptomic sequencing (RNA-Seq) analysis to profile gene response pathways in porcine monocytic cells polarized to typical activation statuses and antiviral states.…”
IMPORTANCE
Activation statuses of monocytic cells, including monocytes, macrophages (Ms), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic, including our focus, PRRSV, which alone causes nearly $800 million economic loss annually in the U.S. swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine the intricate interaction of viral infection with activation statuses and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.
Non‐coding RNA 886 (nc886/VTRNA2‐1) is a Pol III transcript and an atypical imprinted gene. Its exact function as a negative regulator of protein kinase R establishes its connection with innate immunity. Studies have shown that nc886 silencing is closely associated with prostate cancer progression. Previous work has constructed a cell model of stable nc886 overexpression (“mimic” or “nc886
+
”) in PC‐3M‐1E8 cell lines (1E8), which are highly bone‐metastatic human prostate cancer cells with low expression of nc886, and cells expressing the mimic were validated to have lower invasive and metastatic abilities than cells expressing the scramble transcript in vitro and in vivo. In this study, we directly injected mimic or scramble cells into the left ventricle of C57BL/C mice, an immunocompetent animal model, to elucidate the immune mechanisms of tumor‐host interactions. Interestingly, we found that tumor cells induced the inflammation of many important organs due to xenogeneic antigen rejection; this inflammation was ultimately repaired by tissue fibrosis after 28 days, except for in the spleen. The reason is that mimic cells, as heterogeneous antigens, are mostly directly recognized by macrophages or T cells in blood, and few mimic cells enter the spleen compared with scramble cells. The induction of splenic macrophage polarization to M2 macrophages by scramble cells is a critical factor in maintaining chronic splenic inflammation. In addition, we recognize that nc886 broadly decreases the expression of some human leukocyte antigen molecules and antigen transporters. This evidence reveals the interesting role of nc886 in regulating tumor cell antigens.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.