Distinct developmental pathways from blood monocytes generate human lung macrophage diversity.

The study of human macrophages and their ontogeny is an important unresolved issue. Here, we use a humanized mouse model expressing human cytokines to dissect the development of lung macrophages from human hematopoiesis in vivo. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) generated three macrophage populations, occupying separate anatomical niches in the lung. Intravascular cell labeling, cell transplantation, and fate-mapping studies established that classical CD14+ blood monocytes derived from HSPCs migrated into lung tissue and gave rise to human interstitial and alveolar macrophages. In contrast, non-classical CD16+ blood monocytes preferentially generated macrophages resident in the lung vasculature (pulmonary intravascular macrophages). Finally, single-cell RNA sequencing defined intermediate differentiation stages in human lung macrophage development from blood monocytes. This study identifies distinct developmental pathways from circulating monocytes to lung macrophages and reveals how cellular origin contributes to human macrophage identity, diversity, and localization in vivo.

Primary Human M2 Macrophage Subtypes Are Distinguishable by Aqueous Metabolite Profiles.

The complexity of macrophage (MΦ) plasticity and polarization states, which include classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ phenotypes, is becoming increasingly appreciated. Within the M2 MΦ polarization state, M2a, M2b, M2c, and M2d MΦ subcategories have been defined based on their expression of specific cell surface receptors, secreted cytokines, and specialized immune effector functions. The importance of immunometabolic networks in mediating the function and regulation of MΦ immune responses is also being increasingly recognized, although the exact mechanisms and extent of metabolic modulation of MΦ subtype phenotypes and functions remain incompletely understood. In this study, proton (1H) nuclear magnetic resonance (NMR) metabolomics was employed to determine the polar metabolomes of M2 MΦ subtypes and to investigate the relationship between aqueous metabolite profiles and M2 MΦ functional phenotypes. Results from this study demonstrate that M2a MΦs are most distinct from M2b, M2c, and M2d MΦ subtypes, and that M2b MΦs display several metabolic traits associated with an M1-like MΦ phenotype. The significance of metabolome differences for metabolites implicated in glycolysis, the tricarboxylic acid (TCA) cycle, phospholipid metabolism, and creatine-phosphocreatine cycling is discussed. Altogether, this study provides biochemical insights into the role of metabolism in mediating the specialized effector functions of distinct M2 MΦ subtypes and supports the concept of a continuum of macrophage activation states rather than two well-separated and functionally distinct M1/M2 MΦ classes, as originally proposed within a classical M1/M2 MΦ framework.

Soybean lectin-triggered IL-6 secretion induces autophagy to kill intracellular mycobacteria through P2RX7 dependent activation of the JAK2/STAT3/Mcl-1 pathway.

Cytokine therapy and cytokine-mediated autophagy have been used as prominent host-directed therapy (HDT) approaches to restrain M. tb growth in the host cell. In the present study, we have dissected the anti-tubercular activity of Soybean lectin (SBL) through cytokine-mediated autophagy induction in differentiated THP-1 (dTHP-1) cells. A significant increase in IL-6 expression was observed in both uninfected and mycobacteria infected dTHP-1 cells through the P2RX7 mediated pathway via PI3K/Akt/CREB-dependent signalling after SBL treatment. Inhibition of IL-6 level using IL-6 neutralizing antibody or associated signalling significantly enhanced the mycobacterial load in SBL-treated dTHP-1 cells. Further, autocrine signalling of IL-6 through its receptor-induced Mcl-1 expression activated autophagy via JAK2/STAT3 pathway, and inhibition of this pathway affected autophagy. Finally, blocking the IL-6-regulated autophagy through NSC 33994 (a JAK2 inhibitor) or S63845 (an Mcl-1 inhibitor) led to a notable increase in intracellular mycobacterial growth in SBL-treated cells. Taken together, these results indicate that SBL interacts with P2RX7 to regulate PI3K/Akt/CREB network to release IL-6 in dTHP-1 cells. The released IL-6, in turn, activates the JAK2/STAT3/Mcl-1 pathway upon interaction with IL-6Rα to modulate autophagy that ultimately controls mycobacterial growth in macrophages.

Random forest and live single-cell metabolomics reveal metabolic profiles of human macrophages upon polarization.

Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single-cell resolution. To address this knowledge gap, an untargeted live single-cell mass spectrometry-based metabolomic profiling coupled with a machine-learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single-cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single-cell metabolomics provided an in-depth profile of the metabolome of primary human M1 and M2 macrophages at the single-cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells.

P2X7 receptor activation leads to NLRP3-independent IL-1ß release by human macrophages.

BACKGROUND: The purinergic receptor P2X7 plays a crucial role in infection, inflammation, and cell death. It is thought that P2X7 receptor stimulation triggers processing and release of the pro-inflammatory cytokine interleukin (IL)-1ß by activation of the NLRP3 inflammasome; however, the underlying mechanisms remain poorly understood. METHODS: Modulation of IL-1ß secretion was studied in THP-1 macrophages. Adenosine 5'-triphosphate (ATP), BzATP, nigericin and pharmacological inhibitors of P2X receptors, inflammatory caspases and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome were used to characterize signaling. RESULTS: In primed macrophages, IL-1ß release was increased after P2X7 receptor activation by ATP and 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP). Pharmacological inhibition or genetic knockout of NLRP3 does not completely inhibit IL-1ß release in TLR2/1-primed macrophages. Increase in extracellular K+ as well as inhibition of caspase-1 or serine proteases maintained IL-1ß release in macrophages stimulated with P2X7 receptor agonists at 50%. CONCLUSIONS: Our findings suggest a previously unrecognized mechanism of P2X7 receptor mediated IL-1ß release and highlight the existence of an NLRP3-independent pathway in human macrophages. Video Abstract.

Development of a quantitative proteomics approach for cyclooxygenases and lipoxygenases in parallel to quantitative oxylipin analysis allowing the comprehensive investigation of the arachidonic acid cascade.

Oxylipins derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of the arachidonic acid (ARA) cascade are essential for the regulation of the inflammatory response and many other physiological functions. Comprehensive analytical methods comprised of oxylipin and protein abundance analysis are required to fully understand mechanisms leading to changes within these pathways. Here, we describe the development of a quantitative multi-omics approach combining liquid chromatography tandem mass spectrometry-based targeted oxylipin metabolomics and proteomics. As the first targeted proteomics method to cover these pathways, it enables the quantitative analysis of all human COX (COX-1 and COX-2) and relevant LOX pathway enzymes (5-LOX, 12-LOX, 15-LOX, 15-LOX-2, and FLAP) in parallel to the analysis of 239 oxylipins with our targeted oxylipin metabolomics method from a single sample. The detailed comparison between MRM3 and classical MRM-based detection in proteomics showed increased selectivity for MRM3, while MRM performed better in terms of sensitivity (LLOQ, 16-122 pM vs. 75-840 pM for the same peptides), linear range (up to 1.5-7.4 µM vs. 4-368 nM), and multiplexing capacities. Thus, the MRM mode was more favorable for this pathway analysis. With this sensitive multi-omics approach, we comprehensively characterized oxylipin and protein patterns in the human monocytic cell line THP-1 and differently polarized primary macrophages. Finally, the quantification of changes in protein and oxylipin levels induced by lipopolysaccharide stimulation and pharmaceutical treatment demonstrates its usefulness to study molecular modes of action involved in the modulation of the ARA cascade.

Silver nanoparticles exert toxic effects in human monocytes and macrophages associated with the disruption of Δψm and release of pro-inflammatory cytokines.

Silver nanoparticles (AgNP) are the most widely produced type of nanoparticles due to their antimicrobial and preservative properties. However, their systemic bioavailability may be considered a potential hazard. When AgNP reach the bloodstream, they interact with the immune cells, contributing to the onset and development of an inflammatory response. Monocytes and macrophages play a pivotal role in our defense system, but the interaction of AgNP with these cells is still not clear. Therefore, the main objective of this work was to assess the cytotoxic and pro-inflammatory effects induced by 5, 10, and 50 nm AgNP coated with polyvinylpyrrolidone (PVP) and citrate, in concentrations that could be attained in vivo (0-25 µg/mL), in human monocytes isolated from human blood and human macrophages derived from a monocytic cell line (THP-1). The effects of PVP and citrate-coated AgNP on cell viability, mitochondrial membrane potential, and cytokines release were evaluated. The results evidenced that AgNP exert strong harmful effects in both monocytes and macrophages, through the establishment of a strong pro-inflammatory response that culminates in cell death. The observed effects were dependent on the AgNP concentration, size and coating, being observed more pronounced cytotoxic effects with smaller PVP coated AgNP. The results showed that human monocytes seem to be more sensitive to AgNP exposure than human macrophages. Considering the increased daily use of AgNP, it is imperative to further explore the adverse outcomes and mechanistic pathways leading to AgNP-induced pro-inflammatory effects to deep insight into the molecular mechanism involved in this effect.

Mapping the mutual transcriptional responses during Candida albicans and human macrophage interactions by dual RNA-sequencing.

Candida albicans is the leading human fungal pathogen that can cause mucosal and systemic fungal infections. Host phagocytes are the primary immune defense against invading fungal pathogens including C. albicans. To better understand the host-pathogen interaction between C. albicans and host phagocytes, we utilized a human macrophage model of THP-1 macrophages and examined the mutual transcriptomic response of C. albicans and host macrophages by dual RNA-sequencing. Both C. albicans and macrophages displayed marked changes in their transcriptional profiles post 2 h coincubation. We show that C. albicans responds to human macrophages differently than its known response to murine macrophages. C. albicans displays upregulation of its translational machinery and downregulation of glyoxylate and tricarboxylic acid (TCA) cycle upon macrophage phagocytosis. C. albicans triggered strong induction of genes associated with cell surface-mediated signaling and proinflammatory response in THP-1 macrophages. Finally, our data reveal that IL-1ß and TNF signaling are central in mounting a proinflammatory response against C. albicans via MAP kinase, and chemokines and cytokines mediated signaling. Overall, current work uncovers the mutual responses of C. albicans and human macrophages towards each other presenting a better understanding of their interaction during C. albicans infections.

SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders.

INTRODUCTION: The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination. OBJECTIVES: To assess (1) the effects of different Spikes on human primary MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation. METHODS: Human primary MΦ were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts. RESULTS: LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation. CONCLUSIONS: Glycosylated Spike per se is not pro-inflammatory for human MΦ, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination.

Effect of erythromycin on the ultrastructure of human macrophages exposed to cigarette smoke extract in vitro.

Macrophages serve an active role in the pathophysiology of chronic obstructive pulmonary disease (COPD). Erythromycin (EM) has been verified as an effective treatment for COPD. However, there are few studies on the effect of EM on the ultrastructure of macrophages exposed to cigarette smoke extract (CSE). In the present study, human macrophages were randomly divided into three groups: The control, CSE and the CSE+EM group, using electron microscopy, the effect of EM was evaluated by comparing the ultrastructural changes between these groups. The macrophages were additionally divided into a further four groups: The control, CSE, CSE+EM 24 h and CSE+EM 48 h groups. The generation of reactive oxygen species (ROS) in each group was evaluated by detecting fluorescence intensity. It was observed that the cellular ultrastructure of the CSE group exhibited abnormal changes, though this effect was reversed back to the level of the control in the CSE+EM group. Compared with the control group, the ROS expression level was significantly increased in the CSE group (P < .05); however, compared with the CSE group, the ROS concentration was decreased in the CSE+EM 24 h (P < .05) and CSE+EM 48 h groups (P < .05), though this was more apparent in the EM 48 h group. It was concluded that EM protects human macrophages against CSE. Moreover, it was hypothesized that EM may reduce the symptoms of patients with COPD by protecting the macrophage ultrastructure from the effects of CSE, resulting in the decreased generation of ROS, inhibiting autophagy and reducing endoplasmic reticulum stress.

Immunomodulatory Agents Combat Multidrug-Resistant Tuberculosis by Improving Antimicrobial Immunity.

BACKGROUND: Multidrug-resistant (MDR) tuberculosis has low treatment success rates, and new treatment strategies are needed. We explored whether treatment with active vitamin D3 (vitD) and phenylbutyrate (PBA) could improve conventional chemotherapy by enhancing immune-mediated eradication of Mycobacterium tuberculosis. METHODS: A clinically relevant model was used consisting of human macrophages infected with M. tuberculosis isolates (n = 15) with different antibiotic resistance profiles. The antimicrobial effect of vitD+PBA, was tested together with rifampicin or isoniazid. Methods included colony-forming units (intracellular bacterial growth), messenger RNA expression analyses (LL-37, ß-defensin, nitric oxide synthase, and dual oxidase 2), RNA interference (LL-37-silencing in primary macrophages), and Western blot analysis and confocal microscopy (LL-37 and LC3 protein expression). RESULTS: VitD+PBA inhibited growth of clinical MDR tuberculosis strains in human macrophages and strengthened intracellular growth inhibition of rifampicin and isoniazid via induction of the antimicrobial peptide LL-37 and LC3-dependent autophagy. Gene silencing of LL-37 expression enhanced MDR tuberculosis growth in vitD+PBA-treated macrophages. The combination of vitD+PBA and isoniazid were as effective in reducing intracellular MDR tuberculosis growth as a >125-fold higher dose of isoniazid alone, suggesting potent additive effects of vitD+PBA with isoniazid. CONCLUSIONS: Immunomodulatory agents that trigger multiple immune pathways can strengthen standard MDR tuberculosis treatment and contribute to next-generation individualized treatment options for patients with difficult-to-treat pulmonary tuberculosis.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

Regulation of human THP-1 macrophage polarization by Trichinella spiralis.

Trichinella spiralis is a foodborne zoonotic nematode, which causes trichinellosis. During the infection, parasite evades the host immune responses by direct and indirect (through excretory-secretory products) contact with host immune cells. One of the main targets for immunomodulation induced by helminths are macrophages. In this study, we examined whether direct contact of different stages of T. spiralis can affect the polarization of human THP-1 macrophages. Co-culture of adult parasite stage and cells in direct contact without LPS addition had a significant impact on TNFα levels. Interestingly, in settings with the addition of LPS, the levels of IL-1ß and TNFα significantly increased in adult parasite and newborn larvae (NBL) but not for muscle larvae (ML). While we tested muscle larvae ESP products to compare its effect with whole ML parasite, we detect an increase of pro-inflammatory cytokines like IL-1ß and TNFα in no LPS conditions. Whereas, muscle larvae ESP significantly suppressed the inflammatory response measured by IL-1ß, TNFα, and IL-6 levels and anti-inflammatory IL-10 compared to LPS control. Our findings indicate the anti-inflammatory potential of T. spiralis muscle larvae excretory-secretory products and propose signaling pathways which might be engaged in the mechanism of how muscle larvae ESP affect human macrophages.

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction.

The nano-sized membrane enclosed extracellular vesicles (EVs) released by virtually all cell types play an essential role in intercellular communication via delivering bio-molecules, such as nucleic acids, proteins, lipids, and other molecules to recipient cells. By mediating an active and steady-state cell-to-cell communication, EVs contribute to regulating and preserving cellular homeostasis. On the other hand, EVs can also spread pathogen-derived molecules during infections, subverting the host immune responses during infections and thus worsening pathophysiological processes. In recent years, the biological functioning of EVs has become a widespread research field in basic and clinical branches of medical sciences due to their potential role in therapeutic applications for several diseases. This review aims to summarize the main recent findings regarding the implication of EVs shed by human macrophages (MΦ-EVs) and how they can modulate the host immune response to control or increase the damage caused by infectious agents. We will also present the methods used to describe MΦ-EVs, as well as the potential of these EVs as disease diagnostic tools for some human pathogens. We believe that an in-depth understanding of the host-pathogen interactions mediated by MΦ-EVs may trigger the development of innovative therapeutic strategies against infectious diseases.

Influence of Secretome of Different Functional Phenotypes of Macrophages on Proliferation, Differentiation, and Collagen-Producing Activity of Dermal Fibroblasts In Vitro.

We studied the effect of conditioned media of GM-CSF-differentiated human macrophages polarized in M1(LPS), M2a(IL-4), M2c(dexamethasone), and M2(low serum) phenotypes on proliferation, differentiation, and collagen-producing activity of dermal fibroblasts. It was found that M1(LPS) and M2a(IL-4) were characterized by moderate influence on functional activity of fibroblasts. At the same time, soluble factors of M2c(dexamethasone) significantly enhanced the proliferative response of fibroblasts, but not their differentiation and type I collagen production. On the contrary, M2(low serum) generated under conditions of growth factors deficiency had a pronounced stimulating effect on the differentiation of fibroblasts and production of type I collagen by these cells, but moderately stimulated the fibroblast proliferation. Thus, the secretory activity of various functional phenotypes of macrophages is an important mechanism of fibrogenesis regulation.

Efferocytosis Modulates Arginase-1 and Tyrosine Kinase Mer Expression in GM-CSF-Differentiated Human Macrophages.

We studied the expression of arginase-1 (Arg1) and tyrosine kinase Mer (MerTK) in GMCSF-differentiated human macrophage populations М0, М1(IFNγ), М2а(IL-4), and М2(low serum) generated under conditions of growth/serum factor deficiency. The maximum relative content of Arg1+ and MerTK+ cells was found in М2 macrophage populations: М2а(IL-4) and М2(low serum). As the uptake of apoptotic cells is the key mechanism of M2 polarization during M2(low serum) generation, we performed a special series of experiments and showed that incubation with allogeneic apoptotic neutrophils significantly increased the percentages of CD206+ macrophages co-expressing Arg1 and MerTK.

Effect of M2 Macrophage-Derived Soluble Factors on Proliferation and Apoptosis of SH-SY5Y Cells.

Macrophages play the key role in the regulation of neuroregeneration. For evaluation of the neuroregenerative potential of M2 macrophages, we studied the effect of macrophages polarized with IL-4 (M2a (IL-4)) and by efferocytosis under conditions of serum deprivation (LS, Low Serum; M2(LS)) on proliferative activity and apoptosis of SH-SY5Y cells under conditions of deficiency of growth/serum factors. Conditioned media of both M2(LS) and M2a(IL-4) stimulated proliferation of SH-SY5Y cells. Moreover, soluble factors of M2(LS) and M2a(IL-4) reduced the degree of early apoptosis of SH-SY5Y cells and the protective effect of M2(LS) was observed at earlier terms of culturing. Our findings suggest that M2 macrophages have high neuroregenerative potential that is mediated through soluble factors and manifests itself both in stimulation of proliferation and inhibition of apoptosis of SH-SY5Y cells.

Nontuberculous Mycobacteria Show Differential Infectivity and Use Phospholipids to Antagonize LL-37.

Comparisons of infectivity among the clinically important nontuberculous mycobacteria (NTM) species have not been explored in great depth. Rapid-growing mycobacteria, including Mycobacterium abscessus and M. porcinum, can cause indolent but progressive lung disease. Slow-growing members of the M. avium complex are the most common group of NTM to cause lung disease, and molecular approaches can now distinguish between several distinct species of M. avium complex including M. intracellulare, M. avium, M. marseillense, and M. chimaera. Differential infectivity among these NTM species may, in part, account for differences in clinical outcomes and response to treatment; thus, knowing the relative infectivity of particular isolates could increase prognostication accuracy and enhance personalized treatment. Using human macrophages, we investigated the infectivity and virulence of nine NTM species, as well as multiple isolates of the same species. We also assessed their capacity to evade killing by the antibacterial peptide cathelicidin (LL-37). We discovered that the ability of different NTM species to infect macrophages varied among the species and among isolates of the same species. Our biochemical assays implicate modified phospholipids, which may include a phosphatidylinositol or cardiolipin backbone, as candidate antagonists of LL-37 antibacterial activity. The high variation in infectivity and virulence of NTM strains suggests that more detailed microbiological and biochemical characterizations are necessary to increase our knowledge of NTM pathogenesis.

Isolation of functional mature peritoneal macrophages from healthy humans.

Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.

Transfection of hard-to-transfect primary human macrophages with Bax siRNA to reverse Resveratrol-induced apoptosis.

Small interfering RNA (siRNA) is a critical loss-of-function tool for elucidating the role of genes in biomedical studies. The effective use of siRNA needs transfection technology that delivers siRNA into the correct location of target cells, especially those which are extremely difficult to transfect. Macrophages, which play an important role in the pathogenesis of many diseases, are known to be extremely hard to transfect. Thus, to elucidate the functions of genes in human macrophage biology, it is essential to devise technology for efficient siRNA transfection. However, a fast and efficient method for siRNA transfection in primary human macrophages has not been reported. The siRNA transfection is a tug-of-war between transfection rate and cytotoxicity. A higher transfection rate is generally accompanied with increased cytotoxicity, therefore, choosing a transfection reagent that limits cell death while maintain a desirable transfection rate is important. In this study, we employed auto-analysis function of the IncuCyte® to devise a fast and cost-saving technology for efficient transfection of adherent cells and particularly human macrophages. We show that DharmaFECT3 transfection reagent from Dharmacon was the most efficient in transfecting primary human monocyte-derived macrophages and PMA-differentiated U937 cells, whereas other transfection reagents tested were cytotoxic. This method exhibited approximately 85% transfection efficiency in human macrophages. Moreover, siRNA silencing of Bax with this technique effectively protected primary human macrophages and PMA-differentiated U937 cells against Resveratrol-induced cell death. In addition, this method inherently takes the balance between transfection rate and cytotoxicity of siRNA transfection reagents into consideration.