Primary Mononuclear Phagocytes Research Articles

Optimization of a lipid nanoparticle-based protocol for RNA transfection into primary mononuclear phagocytes.

The effective delivery of synthetic RNA into mononuclear phagocytes is a prerequisite for experimental research and therapeutic development. However, traditional methods are highly ineffective and toxic for these cells. Here, we aimed to optimize a transfection protocol for primary bone marrow-derived phagocytes, specifically dendritic cells and macrophages, using lipid nanoparticles generated by microfluidics. Our results show that a lipid mixture similar to that used in Moderna's COVID-19 messenger RNA vaccine outperforms the others tested. Improved messenger RNA transfection can be achieved by replacing uridine with methylpseudouridine but not methoxyuridine, which interferes with transfection. The addition of diphenyleneiodonium or apocynin can enhance transfection in a cell type-dependent manner without adverse effects, while apolipoprotein E provides no added value. These optimized transfection conditions can also be used for microRNA agonists and antagonists. In sum, this study offers a straightforward, highly efficient, reproducible, and nontoxic protocol to deliver RNA into different primary mononuclear phagocytes in culture.

Localization of the LRBA protein in the endomembrane system of HELA cells and primary mononuclear phagocytes

Localization of the LRBA protein in the endomembrane system of HELA cells and primary mononuclear phagocytes

CpG oligonucleotides bind TLR9 and RRM-Containing proteins in Atlantic Salmon (Salmo salar)

BackgroundBacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates.ResultsIn the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes.ConclusionThe presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.

Kinase Inhibitors Reduce TNF-Alpha Over-Production in Monocytes From Fanconi Anemia Group A Patients

Abstract 2409Fanconi Anemia (FA) is a chromosomal instability syndrome with hypersensitivity to alkylating agents as principal diagnostic feature. Many laboratories have demonstrated the involvement of FA proteins in DNA repair mechanisms. Recent work has also demonstrated other functions of some FA proteins suggesting that they play alternative roles in other regulatory pathways, particularly those that influence hematopoiesis.Eighty percent of FA patients develop bone marrow failure with a high incidence of evolution in myelodysplasia and/or acute leukemia. Each of these abnormalities has been related to TNF-alpha hypersensitivity in the stem and progenitor cell pools and the toll-like receptor dependent overproduction of TNF-alpha by FA macrophages. The TNF-hypersensitive phenotype involves at least two kinases, PKR and ASK1, each of which is hyperactivated in FA cells and induce apoptotic responses both in ground state and after TNF-alpha and IFN-gamma stimulation. Recent evidence showed that R848 (a TLR8 ligand) and endotoxin (LPS, a TLR4 ligand)-induced TNF-alpha gene expression in Fancc-deficient mononuclear phagocytes cells is inhibited by kinase inhibitors dasatinib and BIRB796 We sought to evaluate the activity of these agents in primary mononuclear phagocytes obtained from children with FA-A. Objectives:To determine whether primary monocytes from the peripheral blood of FANCA-deficient patients: (a) exhibit the TNF-overproduction phenotype in response to LPS and the TLR8 ligand R848, and (b) respond to dasatinib and BIRB796 by suppressing TNF-production. Methods:Six FA patients with mild to severe marrow failure on no treatment were included in this study. Healthy subjects were recruited as normal controls that were run in parallel in each case. CD14+ monocytes freshly isolated from peripheral blood were cultured for 24 hours with LPS and R848 with or without dasatinib or BIRB796. Supernatant media were collected and frozen at −80 degrees. After thawing the samples, TNF-alpha content was quantified by ELISA. Results:Baseline TNF-alpha concentration (without any TLR stimulation) was higher in FA patients than control. After LPS or R848 stimulation FA-A monocytes produced substantially more TNF-alpha than did the control samples. Both dasatinib and BIRB796 suppressed TLR-induced (both LPS and R848) TNF-alpha production. Specifically, with R848 as the agonist, BIRB 769 suppressed TNF-alpha production by 60% and dasatinib by 42%. Both inhibitors were even more potent in suppressing LPS induced TNF-alpha expression as both reduced TNF-alpha by 75%. In the absence of TLR stimulation, the presence of BIRB or dasatinib in culture reduced TNF-alpha by >50% compared to baseline in patient samples. The inhibitory effect of kinase inhibitors was observed also in the normal control. Conclusions:These findings: (a) demonstrate for the first time that TLR-induced TNF-alpha gene expression in primary FANCA deficient mononuclear phagocytes is aberrantly regulated and (b) that in FANCA-deficient macrophages the TNF-alpha overproduction phenotype can be controlled by therapeutically achievable doses of BIRB796 and dasatinib. In addition (c), since both agents function in large part to suppress p38 MAPK activation future, our data point to the biochemical roles played by FANCA in modulating upstream pathways that govern p38 activation. Moreover (d), given that in FA patients, TNF hypersensitive stem cells are over-exposed to TNF-alpha, particularly during inflammatory events and that exposure to TNF was shown not only to suppress hematopoiesis in FA but also to favor the emergence of neoplastic clones, these results point to these two agents as potential candidates for preclinical trials seeking to enhance hematopoiesis and suppress clonal evolution. Disclosures:No relevant conflicts of interest to declare.

Quantitative measurement of anthrax toxin receptor messenger RNA in primary mononuclear phagocytes

Two anthrax toxin receptors have been identified, tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). Both receptors have been shown to be capable of facilitating the entry of Bacillus anthracis exotoxins into the cytosol of susceptible cells. The levels of TEM8 and CMG2 RNA transcripts present in human primary macrophages derived from multiple unrelated donors and primary mouse macrophages have not been well described. In this communication, we examined the expression of mRNA transcripts of TEM8 and CMG2 in primary human and mouse macrophages and mouse tissues by standard and quantitative real-time RT-PCR. Our results indicate that CMG2 transcripts are preferentially expressed over TEM8 transcripts in primary human and mouse macrophages as compared to immortalized cell lines.

Transactivation of human immunodeficiency virus-1 in T-cells by Mycobacterium tuberculosis-infected mononuclear phagocytes

Worldwide, tuberculosis (TB) is the most frequent coinfection with human immunodeficiency virus-1 (HIV-1), and active TB enhances the progression of HIV-1 disease in dually infected subjects. In the microenvironment of Mycobacterium tuberculosis (MTB)–infected foci, where HIV-1–infected CD4 T-cells come into contact with MTB-infected macrophages, the direct interaction of the two cell types in the activation of latent HIV-1 may be important. In this study we sought to determine whether MTB-infected human primary mononuclear phagocytes—namely, alveolar macrophages (AMs) and their less mature blood precursors, monocytes—activate HIV-1 in a T-cell line stably transfected with an HIV-1 long terminal repeat (LTR) reporter construct (1G5 cells) and induce HIV-1 expression in T-cells from HIV-1–infected subjects. MTB-infected monocytes and AMs, and not Mycobacterium avium–infected cells, activated HIV-1 LTR in 1G5 cells in the presence and absence of HIV-1 tat. Transactivation of HIV-1 LTR by MTB-infected mononuclear phagocytes was mediated mainly by tumor necrosis factor-α. In AMs, but not monocytes, membrane tumor necrosis factor-α contributed to transactivation of HIV-1 LTR. MTB-infected MNs from 60% of HIV-infected subjects induced HIV-1 LTR in 1G5 cells as well. Furthermore, HIV-1 transcription was induced in autologous T-cells from 30% of the HIV-1–infected subjects. We therefore conclude that MTB-infected mononuclear phagocytes can transactivate HIV-1 in CD4 cells. Transactivation of latent HIV-1 in CD4 T-cells by MTB-infected mononuclear phagocytes may in part be responsible for increased HIV activity at sites of MTB infection during dual infection in vivo.

Recombinant IFN-α (2b) Increases the Expression of Apoptosis Receptor CD95 and Chemokine Receptors CCR1 and CCR3 in Monocytoid Cells

AbstractIFN-α-2b, known as potent immune modulator, can either inhibit or enhance immune cell activity within the tightly regulated microenvironment of inflammation, depending upon the concentration of the cytokine and the activation stage of the cell. Chemokine receptors, which not only mediate chemotaxis of immune cells to the site of inflammation but also affect cellular activation by transferring corresponding signals, represent yet another level of immune regulation. Here we demonstrate that IFN-α increases the expression of CCR1 and CCR3 in primary mononuclear phagocytes, as well as in the monocytoid cell line U937. Enhanced receptor mRNA expression correlated with functional readouts such as increased intracellular calcium mobilization and cell migration in response to ligands. Expression of CCR2b, CCR4, CCR5, and CXCR4 was unchanged or decreased after IFN-α treatment. These observations indicate a differentially regulated cellular signaling relationship of IFN-α pathways and chemokine receptor expression. We also provide evidence that, under these conditions, IFN-α treatment increased the expression of CD95 (Fas, Apo1), resulting in enhanced susceptibility to apoptosis. Taken together, these data add important information for the rational application of IFN-α (2b) in immune and cancer therapies.

Tropism for primary monocytes and for monocytoid cell lines are separate features of HIV-1 variants.

We have analyzed the phenotype of five human monocytoid cell lines (U-937, THP-1, RC.2A, Mono Mac 6, and DD) and their capacity to support replication of HIV-1 strains with known tropism for T lymphocytes and mononuclear phagocytes. HIV-1 Ba-L, a prototype monocytotropic HIV-1 strain, was unable to establish continuous replication in any of the cell lines used. In contrast, the lymphotropic strain IIIB could replicate in all CD4-positive cell lines. We conclude that the capacity of HIV-1 isolates to replicate in established human monocytoid malignant cells does not correlate with the tropism of the virus for primary mononuclear phagocytes. Since monocytoid cell lines do not distinguish HIV-1 variants tropic for mononuclear phagocytes, we suggest the use of primary cells for studies of HIV-1 tropism.

Anti-HIV-1 activity of 3′-azido-3′-deoxythymidine (AZT) in primary mononuclear phagocytes

Conflicting data have been reported on ability of 3′-azido-3′-deoxythymidine (AZT) to protect mononuclear phagocytes from HIV-1 infection. We compared the antiviral potency of AZT in three types of primary human mononuclear phagocytes: peripheral blood monocytes, monocyte-derived macrophages (in vitro differentiated) and alveolar macrophages (in vivo differentiated). To establish highly-productive virus infection, purified cells (> 99%) from healthy donors were challenged with the macrophage-tropic HTLV-III Ba-L strain at input multiplicities ranging from 0.05 to 20 TCID 50 per cell. AZT (0.1 nM–10 μM) was added immediately after infection and either continued for the duration of the experiment or stopped 1–7 days after infection. The kinetics of HIV-1 Ba-L replication were assessed by measuring p24 antigen production on days 4–28 post-infection. Continuous treatment with AZT reproducibly inhibited viral replication in a concentration-dependent manner in all three cell types. The IC 90 of AZT was 0.04 μM in blood monocytes, 0.009 μM in monocyte-derived macrophages, and 0.0001 μM in alveolar macrophages (mean of 3–4 donors for each cell type). AZT was not cytotoxic at < 10 μM as assessed by cell viability, cell protein, and interferon-γ-activated H 2O 2-release. In experiments in which AZT treatment was stopped after infection, viral replication resumed after a lag of 7–14 days and increased exponentially toward control levels. This occurred despite initial inhibition of virus production to below the limit of p24 detection (∼ 50 pg/ml). These results indicate that AZT is a potent inhibitor of HIV-1 replication in primary mononuclear phagocytes regardless of the stage of cell differentiation, and that AZT is most active in tissue (alveolar) macrophages. AZT does not irreversibly block infection of mononuclear phagocytes, however, as viral replication resumes after removal of AZT.

In vitro effect of transforming growth factor-beta on progression of HIV-1 infection in primary mononuclear phagocytes.

In vitro-differentiated monocytes can be infected with the monocytotropic isolate of HIV-1/ADA. The infection is characterized by formation of giant cells and production of virus that can be found in cell supernatants or cell-associated. In this study, we demonstrate that the above described parameters of infection can be enhanced by a factor present in acidified M phi supernatants, suggesting that it might be transforming growth factor beta-1 (TGF-beta 1). When recombinant or purified TGF-beta were examined, similar activities were detected. This effect apparently is not because of changes in the cellular phenotype that could favor infection. The effect of TGF-beta is exerted on cells once infection is established or on cells with active virus production. The activity can be also demonstrated using U-937 cells.

HIV-1 Infection Does Not Induce Tumor Necrosis Factor-?? or Interferon-?? Gene Transcription

An early host defense against infection by RNA or DNA viruses is the induction, within infected cells, of tumor necrosis factor-alpha (TNF-alpha) gene transcription. The protein product of the TNF-alpha gene alone, or together with different types of interferons, inhibits viral propagation in diverse cell types. In this study, the effect of acute and chronic human immunodeficiency virus type 1 (HIV-1) infection on the transcription of the TNF-alpha and interferon-beta (IFN-beta) genes was examined in susceptible monocyte and T-cell lines as well as in primary human mononuclear phagocytes. Although Sendai virus, a prototypic inducer of TNF-alpha and IFN-beta mRNA, induced the transcription of both genes in the monocyte cell lines and TNF-alpha in the T-cell line and in primary mononuclear phagocytes, transcription of these genes was not inducible by HIV-1. Therefore, HIV-1 was able to infect these cells without triggering the transcription of genes encoding proteins important in immediate antiviral cellular defenses. These results may explain in part how HIV-1 is able to establish persistent intracellular infections and escape acute host responses that have evolved to combat viral infection.

Cytokines Alter Production of HIV-1 from Primary Mononuclear Phagocytes

Some strains of human immunodeficiency virus type 1 (HIV-1) can infect primary monocytes and monocyte-derived macrophages in vitro. In this report, the effect of cytokines on the production of one of these strains that shows a tropism for mononuclear phagocytes, designated HIV-1JR-FL, was studied. Primary peripheral blood mononuclear phagocytes infected with HIV-1JR-FL were treated with the hematopoietic factors: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), macrophage colony-stimulating factor (M-CSF), and gamma-interferon (gamma-IFN). The M-CSF, GM-CSF, IL-3, and gamma-IFN were able to alter HIV-1 production under different conditions.

Cytokines alter production of HIV-1 from primary mononuclear phagocytes.

Some strains of human immunodeficiency virus type 1 (HIV-1) can infect primary monocytes and monocyte-derived macrophages in vitro. In this report, the effect of cytokines on the production of one of these strains that shows a tropism for mononuclear phagocytes, designated HIV-1JR-FL, was studied. Primary peripheral blood mononuclear phagocytes infected with HIV-1JR-FL were treated with the hematopoietic factors: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), macrophage colony-stimulating factor (M-CSF), and gamma-interferon (gamma-IFN). The M-CSF, GM-CSF, IL-3, and gamma-IFN were able to alter HIV-1 production under different conditions.