Human Macrophage Function Research Articles

Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions.

Macrophages fight infection and ensure tissue repair, often operating at nutrient-poor wound sites. We investigated the ability of human macrophages to metabolize glycogen. We observed that the cytokines GM-CSF and M-CSF plus IL-4 induced glycogenesis and the accumulation of glycogen by monocyte-derived macrophages. Glyconeogenesis occurs in cells cultured in the presence of the inflammatory cytokines GM-CSF and IFNγ (M1 cells), via phosphoenolpyruvate carboxykinase 2 (PCK2) and fructose-1,6-bisphosphatase 1 (FBP1). Enzyme inhibition with drugs or gene silencing techniques and 13C-tracing demonstrate that glutamine (metabolized by the TCA cycle), lactic acid, and glycerol were substrates of glyconeogenesis only in M1 cells. Tumor-associated macrophages (TAMs) also store glycogen and can perform glyconeogenesis. Finally, macrophage glycogenolysis and the pentose phosphate pathway (PPP) support cytokine secretion and phagocytosis regardless of the availability of extracellular glucose. Thus, glycogen metabolism supports the functions of human M1 and M2 cells, with inflammatory M1 cells displaying a possible dependence on glyconeogenesis.

Monocyte and macrophage profiles in patients with inherited long-chain fatty acid oxidation disorders

Patients with inherited disorders of the long-chain fatty acid oxidation (lcFAO) machinery present with a heterogeneous profile of disease manifestations and aggravation of symptoms is often triggered by inflammatory activation. Monocytes and macrophages are innate immune cells that play a major role in the onset and resolution of inflammation. These cells undergo metabolic rewiring upon activation including the regulation of the FAO rate. The rewiring of FAO and the effect of lcFAO disorders (lcFAOD) on human monocyte and macrophage phenotype and function remain largely unknown. Here, we performed extensive phenotyping of circulating monocytes and analyzed plasma cytokine levels in 11 lcFAOD patients and 11 matched control subjects. In patients with lcFAOD, we observed induced plasma levels of the inflammatory cytokines IL-1β and IL-6, and enhanced CD206 and CD62L surface marker expression in circulating monocyte subsets. To mimic the most common lcFAOD very-long-chain acyl-CoA dehydrogenase disorder (VLCADD), we used siRNA-mediated knockdown of the ACADVL gene (encoding VLCAD) in macrophages derived from healthy volunteers. Hereby, we found that siVLCAD affected IL-4-induced alternative macrophage activation while leaving LPS responses and cellular metabolism intact. In the same line, monocyte-derived macrophages from lcFAOD patients had elevated levels of the IL-4-induced alternative macrophage markers CD206 and CD200R. Still, they did not show major metabolic defects or changes in the LPS-induced inflammatory response. Our results indicate that monocytes and macrophages from lcFAOD patients present no major inflammatory or metabolic differences and show that IL-4-induced surface markers are intertwined with lcFAO in human macrophages.

Overexpressing miR-222-3p in cultured <i>Mycobacterium Tuberculosis</i>-infected macrophages does not affect their bacteriostatic activity

Tuberculosis, a disease caused by the bacterium Mycobacterium tuberculosis, is a major public health concern. Innate and adaptive immunity provide robust defense against pathogens. However, M. tuberculosis, which co-evolved with humans, has acquired many mechanisms to evade the immune response and ensure its intracellular existence and long-term survival within the host. Moreover, emerging evidence suggests that this secretive bacterium can alter expression of regulatory noncoding RNAs (including microRNAs), leading to dysregulation of biological processes underlying tuberculosis pathogenesis. For example, miR-222-3p has been shown to regulate the functional reprogramming of macrophages and is involved in the regulation of host innate immunity. Previously, we demonstrated the important role of miR-222-3p as a biological marker of tuberculosis activity. To confirm their biological targets and understand their role in the pathogenesis of tuberculosis, many research groups are working to establish functional relationships between miRNA expression under different conditions and their actual biological action using molecular biology and bioinformatics methods. In the present study, we demonstrated the effect of miR-222-3p overexpression on several functions of human macrophages of monocytic origin activated with M. tuberculosis antigens in in vitro culture. Specifically, we found that miR-222-3p overexpression significantly decreased IL-6 and IFNγ expression and increased IL-1β and cxcl10 expression in cultures of uninfected macrophages. Infected macrophages overexpressing miR-222-3p were characterized by increased NF-κB and IL-6 expression, as were infected macrophages without transfection. Another important finding was that miR-222-3p overexpression caused a small but significant increase in reactive nitrogen species production by infected macrophages, but did not affect their bacteriostatic activity against M. tuberculosis. Elucidating the functions of different microRNAs in regulating different pathogenic pathways in TB may lead to discovering new therapeutic targets. The detailed study of microRNAs that regulate immune-associated pathways will be useful for the design of miRNA mimetic molecules, either as inhibitors or as activators. Immune effects induced by miRNA drugs are currently a major challenge for miRNA therapeutics.

Abstract 6538: Modulation of human macrophage differentiation, phenotype and function in vitro as a strategy to characterize novel tumor microenvironment modulators

Abstract Macrophage infiltration of the tumor microenvironment is a key process that can determine the success or failure of anti-tumor therapies. Thus, many immunooncology therapies are being developed to modulate the macrophage differentiation program towards the anti-tumor M1 subset, while hindering the M2 subset associated with tumor progression. Monocyte-derived macrophages comprise a crucial in vitro cellular tool to evaluate the plasticity potential between the different polarization states. Several hurdles like their in vitro adherence, low cell number yield and the donor-to-donor variability pose technical challenges when using in vitro differentiated macrophages for compound screening and titration or functional assays. At Reaction Biology we have established human macrophage assay pipelines to evaluate the novel macrophage polarization compounds in vitro. By isolating monocytes from frozen PBMC stocks it is possible to have access to the same donor or group of donors PBMC for repetitive analysis. We have developed a differentiation protocol that allows us to use different plate format from 6-well to 48 well plates, and thus increase and modulate the throughput according to the required readout. The effect of therapeutic compounds can be analyzed by flow cytometry. Our data shows that human primary monocytes grown under M1 polarizing conditions express high levels of CD86 and CD80 in contrast to those cultivated under M2 polarizing conditions, with high expression of CD163, CD206, and CD209. Cell culture supernatant can be used to determine cytokine secretion. The functionality of the different macrophage subtypes can be further investigated using a pH-sensitive phagocytosis tracker, like pHRodo-Zymosan or pHRodo-Ecoli, which shows differential uptake according to differentiation profile. Moreover, treated macrophages can be used in coculture with T cells to investigate their capability to foster or inhibit an immune response. Our data shows the value of monocyte-derived macrophage assays in the evaluation and efficacy assessment of TME modulators. Citation Format: Carla N. Castro, Tamara Sahner, Cynthia Obodozie, Philipp Metzger, Holger Weber. Modulation of human macrophage differentiation, phenotype and function in vitro as a strategy to characterize novel tumor microenvironment modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6538.

IMMU-28. TREM2 INHIBITION REPROGRAMS MYELOID CELLS TO STIMULATE ANTI-TUMOR IMMUNITY THROUGH BOTH DIRECT AND INDIRECT MECHANISMS

Abstract The prevailing view is that myeloid cells—macrophages and microglia—in the tumor microenvironment (TME) are immunosuppressive and promote glioblastoma (GBM) progression. However, myeloid cells have the functional plasticity to restrict or support tumor cell growth. TREM2 plays important roles in brain microglial function in neurodegenerative diseases, but the functional role of TREM2 in the GBM TME and whether TREM2 represents a meaningful therapeutic target remain largely unknown. To address these questions, we investigated the immune and tumor cell consequences of TREM2 inhibition by multiple orthogonal approaches—transgenic mice, antisense oligonucleotides, and blocking antibodies—in immunocompetent preclinical models (SB28, NPA C54B) and human GBM samples through bulk and single cell RNA-sequencing, flow cytometry, biochemistry, and functional assays. We found TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors, and that high TREM2 expression correlates with poor prognosis in GBM patients. TREM2 loss of function in human macrophages and mouse myeloid cells increased IFNγ-induced immunoactivation and proinflammatory polarization. Conditioned media from TREM2 deficient human macrophages directly killed patient-derived GBM tumor cells, and biochemical analysis identified TNF and other putative tumoricidal proteins in this conditioned media. In mouse GBM models, mice with chronic and acute Trem2 loss of function demonstrated increased tumor cell death and increased animal survival. Trem2 inhibition reprogrammed myeloid cell phenotypes and increased PD-1+CD8+ T cells in the TME. Remarkably, Trem2 deficiency enhanced the effectiveness of anti-PD-1 treatment in vivo. Trem2 inhibition suppresses tumor cell growth through direct and indirect immune cell mechanisms--by reshaping myeloid subset and T cell functions. Thus, Trem2 inhibitory strategies, in combination with immune checkpoint blockade, may represent an attractive new approach for GBM therapy.

Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.

Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria.

Phenotyping of Macrophages in Human Immune System Mice.

Human immune system mice, also referred to as humanized mice, are a major research tool for the in vivo study of human immune system function. Upon reconstitution with human hematopoietic stem cells, all major human leukocyte populations develop in immunodeficient mice and can be detected in peripheral blood as well as in lymphatic and nonlymphatic tissue. This includes human macrophages that are intrinsically difficult to study from humans due to their organ-resident nature. In the following chapter, we provide a detailed protocol for generation of human immune system mice. We suggest that these mice are a suitable model to study human macrophage function in vivo.

A Model of iPSC-Derived Macrophages with TNFAIP3 Overexpression Reveals the Peculiarities of TNFAIP3 Protein Expression and Function in Human Macrophages.

Macrophages play a crucial role in the development and control of inflammation. Understanding the mechanisms balancing macrophage inflammatory activity is important to develop new strategies for treating inflammation-related diseases. TNF-α-induced protein 3 (TNFAIP3, A20) is a negative regulator of intracellular inflammatory cascades; its deficiency induces hyper-inflammatory reactions. Whether A20 overexpression can dampen macrophage inflammatory response remains unclear. Here, we generated human-induced pluripotent stem cells with tetracycline-inducible A20 expression and differentiated them into macrophages (A20-iMacs). A20-iMacs displayed morphology, phenotype, and phagocytic activity typical of macrophages, and they displayed upregulated A20 expression in response to doxycycline. A20 overexpression dampened the A20-iMac response to TNF-α, as shown by a decreased expression of IL1B and IL6 mRNA. A dynamic analysis of A20 expression following the generation of A20-iMacs and control iMacs showed that the expression declined in iMacs and that iMacs expressed a lower molecular weight form of the A20 protein (~70 kDa) compared with less differentiated cells (~90 kDa). A low-level expression of A20 and the predominance of a low-molecular-weight A20 form were also characteristic of monocyte-derived macrophages. The study for the first time developed a model for generating macrophages with an inducible expression of a target gene and identified the peculiarities of A20 expression in macrophages that likely underlie macrophage preparedness for inflammatory reactivity. It also suggested the possibility of mitigating inflammatory macrophage responses via A20 overexpression.

CD300f signalling induces inhibitory human monocytes/macrophages

The CD300 glycoproteins are a family of related leucocyte surface molecules that regulate the immune response via their paired triggering and inhibitory receptors. Here we studied CD300f, an apoptotic cell receptor, and how it modulates the function of human monocytes and macrophages. We showed that CD300f signalling by crosslinking with anti-CD300f mAb (DCR-2) suppressed monocytes causing upregulation of the inhibitory molecule, CD274 (PD-L1) and their inhibition of T cell proliferation. Furthermore, CD300f signalling drove macrophages preferentially towards M2-type with upregulation of CD274, which was further enhanced by IL-4. CD300f signalling activates the PI3K/Akt pathway in monocytes. Inhibition of PI3K/Akt signalling resulting from CD300f crosslinking leads to downregulation of CD274 expression on monocytes. These findings highlight the potential use of CD300f blockade in cancer immune therapy to target immune suppressive macrophages in the tumour microenvironment, a known resistance mechanism to PD-1/PD-L1 checkpoint inhibitors.

TREM2 inhibition triggers antitumor cell activity of myeloid cells in glioblastoma.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays important roles in brain microglial function in neurodegenerative diseases, but the role of TREM2 in the GBM TME has not been examined. Here, we found that TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors and that high TREM2 expression correlates with poor prognosis in patients with GBM. TREM2 loss of function in human macrophages and mouse myeloid cells increased interferon-γ-induced immunoactivation, proinflammatory polarization, and tumoricidal capacity. In orthotopic mouse GBM models, mice with chronic and acute Trem2 loss of function exhibited decreased tumor growth and increased survival. Trem2 inhibition reprogrammed myeloid phenotypes and increased programmed cell death protein 1 (PD-1)+CD8+ T cells in the TME. Last, Trem2 deficiency enhanced the effectiveness of anti-PD-1 treatment, which may represent a therapeutic strategy for patients with GBM.

Palmitic Acid-Induced Long Noncoding RNA PARAIL Regulates Inflammation via Interaction With RNA-Binding Protein ELAVL1 in Monocytes and Macrophages.

Obesity and diabetes are associated with elevated free fatty acids like palmitic acid (PA), which promote chronic inflammation and impaired inflammation resolution associated with cardiometabolic disorders. Long noncoding RNAs (lncRNAs) are implicated in inflammatory processes; however, their roles in PA-regulated inflammation and resolution are unclear. We performed RNA-sequencing analysis to identify PA-regulated coding genes and novel lncRNAs in CD14+ monocytes from healthy volunteers. We investigated the regulation and function of an uncharacterized PA-induced lncRNA PARAIL (PA-regulated anti-inflammatory lncRNA). We examined its role in inflammation resolution by employing knockdown and overexpression strategies in human and mouse macrophages. We also used RNA pulldown coupled with mass spectrometry to identify PARAIL interacting nuclear proteins and their mechanistic involvement in PARAIL functions in human macrophages. Treatment of human CD14+ monocytes with PA-induced several lncRNAs and genes associated with inflammatory phenotype. PA strongly induced lncRNA PARAIL expressed near RIPK2. PARAIL was also induced by cytokines and infectious agents in human monocytes/macrophages and was regulated by NF-κB (nuclear factor-kappa B). Time course studies showed PARAIL was induced during inflammation resolution phase in PA-treated macrophages. PARAIL knockdown with antisense oligonucleotides upregulated key inflammatory genes and vice versa with PARAIL overexpression. We found that PARAIL interacts with ELAVL1 (ELAV-like RNA-binding protein 1) protein via adenylate/uridylate-rich elements (AU-rich elements; AREs). ELAVL1 knockdown inhibited the anti-inflammatory functions of PARAIL. Moreover, PARAIL knockdown increased cytosolic localization of ELAVL1 and increased the stability of ARE-containing inflammatory genes. Mouse orthologous Parail was downregulated in macrophages from mice with diabetes and atherosclerosis. Parail overexpression attenuated proinflammatory genes in mouse macrophages. Upregulation of PARAIL under acute inflammatory conditions contributes to proresolution mechanisms via PARAIL-ELAVL1 interactions. Conversely, PARAIL downregulation in cardiometabolic diseases enhances ELAVL1 function and impairs inflammation resolution to further augment inflammation. Thus, inflammation-resolving lncRNAs like PARAIL represent novel targets to combat inflammatory cardiometabolic diseases.

The Effects of Ginkgo biloba Extract on Autophagy in Human Macrophages Stimulated by Cigarette Smoke Extract.

To investigate the effects of Ginkgo biloba extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE). The human monocyte cell line U937 was cultured in vitro, and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting. U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant (p < 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group (p < 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased (p < 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group. GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.

TRNA-like Transcripts from the NEAT1-MALAT1 Genomic Region Critically Influence Human Innate Immunity and Macrophage Functions.

The evolutionary conserved NEAT1-MALAT1 gene cluster generates large noncoding transcripts remaining nuclear, while tRNA-like transcripts (mascRNA, menRNA) enzymatically generated from these precursors translocate to the cytosol. Whereas functions have been assigned to the nuclear transcripts, data on biological functions of the small cytosolic transcripts are sparse. We previously found NEAT1-/- and MALAT1-/- mice to display massive atherosclerosis and vascular inflammation. Here, employing selective targeted disruption of menRNA or mascRNA, we investigate the tRNA-like molecules as critical components of innate immunity. CRISPR-generated human ΔmascRNA and ΔmenRNA monocytes/macrophages display defective innate immune sensing, loss of cytokine control, imbalance of growth/angiogenic factor expression impacting upon angiogenesis, and altered cell-cell interaction systems. Antiviral response, foam cell formation/oxLDL uptake, and M1/M2 polarization are defective in ΔmascRNA/ΔmenRNA macrophages, defining first biological functions of menRNA and describing new functions of mascRNA. menRNA and mascRNA represent novel components of innate immunity arising from the noncoding genome. They appear as prototypes of a new class of noncoding RNAs distinct from others (miRNAs, siRNAs) by biosynthetic pathway and intracellular kinetics. Their NEAT1-MALAT1 region of origin appears as archetype of a functionally highly integrated RNA processing system.

IMMU-35. TREM2 RESTRAINS ANTI-TUMOR CELL ACTIVITY OF MYELOID CELLS IN GLIOBLASTOMA

Abstract Tumor-associated myeloid cell populations occupy a major part of the glioblastoma (GBM) tumor microenvironment (TME). The prevailing view is that myeloid cells in the TME are immunosuppressive and promote GBM tumor progression. However, myeloid cells have the functional plasticity to either restrict or support tumor cell growth. TREM2 has been shown to alter the myeloid cell landscape in cancers arising in the body and plays important roles in brain microglial function in neurodegenerative diseases. But the role of TREM2 in the GBM TME and specifically in myeloid cell function has not been examined. Here we found that TREM2 is highly expressed in myeloid subsets, including macrophages and microglia in human and mouse GBM tumors, and that high TREM2 expression is associated with poor prognosis in GBM patients. TREM2 loss of function in human macrophages and mouse myeloid cells increased tumoricidal capacity in vitro using patient-derived glioblastoma stem cells and mouse glioblastoma cells. Accordingly, we found TREM2 in myeloid cells restricts proinflammatory polarization in both LPS-induced innate and IFNγ-induced adaptive immunity in vitro, mainly through the inhibition of NFκB and MAPK p38 signaling pathways. Orthotopic injection of mouse glioblastoma cells into TREM2 knockout mice increased animal survival compared to littermate wildtype mice. In addition, co-implantation of TREM2 knockdown macrophages or microglia with mouse glioblastoma cells in brains of immunocompetent mice increased animal survival compared to co-implantation of control myeloid cells or injection of tumor cells alone, suggesting an important role for TREM2 in myeloid cells in GBM growth in vivo. Together, these data indicate that TREM2 operates by restricting the anti-tumor and proinflammatory function of myeloid cells in GBM and that inhibition of the TREM2 pathway may represent a potential therapeutic strategy for these patients.

Neuropeptide Y Promotes Human M2 Macrophage Polarization and Enhances p62/SQSTM1-Dependent Autophagy and NRF2 Activation.

Neuropeptide Y (NPY) is an abundantly expressed peptide capable of modulating innate and adaptive immune responses and regulating chemotaxis and cytokine secretion by macrophages. Abnormal regulation of NPY is involved in the development of atherosclerosis. The inflammatory infiltrate within atherosclerotic plaque is characterized by accumulation of macrophages, which are subject to reprogram their phenotypes in response to environmental signals. Macrophage number and phenotype influence plaque fate. Here, we investigated the effect of NPY on the changes in phenotype and functions of human macrophages, from the pro-inflammatory phenotype M1 to the reparative M2, indicative of atherosclerosis regression or stabilization. Human monocytes were differentiated in vitro into macrophages with M-CSF (M0) and polarized towards an M1 phenotype with IFN-γ plus LPS M(IFN-γ/LPS) or M2 with IL-10 (M IL-10) and further challenged with NPY (10-7-10-9 M) for 8-36 h. Cell phenotype and functions were analyzed by immunofluorescence and immunochemical analyses. NPY affected macrophage surface markers and secretome profile expression, thus shifting macrophages toward an M2-like phenotype. NPY also prevented the impairment of endocytosis triggered by the oxysterol 7-keto-cholesterol (7KC) and prevented 7KC-induced foam cell formation by reducing the lipid droplet accumulation in M0 macrophages. NPY-treated M0 macrophages enhanced the autophagosome formation by upregulating the cell content of the autophagy markers LC3-II and p62-SQSTM1, increased activation of the anti-oxidative transcription factor NRF2 (NF-E2-related factor 2), and subsequently induced its target gene HMOX1 that encodes heme oxygenase-1. Our findings indicate that NPY has a cytoprotective effect with respect to the progression of the inflammatory pathway, both enhancing p62/SQSTM1-dependent autophagy and the NRF2-antioxidant signaling pathway in macrophages. NPY signaling may have a crucial role in tissue homeostasis in host inflammatory responses through the regulation of macrophage balance and functions within atherosclerosis.

Effect of Sublethal Concentration of Malathion Insecticide on Innate Immune System, Immune Function and Hemocytes of Adult Drosophila melanogaster

Invertebrate organisms mostly have innate immune system. Hemocytes are the immune cells of the innate immunity in invertebrates. Our study was designed to observe the effect of malathion insecticide on the innate immune system, immune function and hemocytes of Drosophila melanogaster. A hemocytometer was used for total count of hemocytes which showed a reduced percentage of live cells (from 91.63% to 44.57%) due to the treatment. For differential count, the hemolymph was spread as a smear on a clean grease free slide. It was observed that the plasmatocyte count considerably decreased under the effect of malathion (from 78% to 44.66%). Lamellocytes also showed a decline in their number (from 18.33% to 12.61%). But, a trivial increase (from 3.67% to 3.98%) in crystal cell count was recorded. Our result suggests that malathion has mostly negative effect on hemocytes. This study could help in understanding of insect defense mechanism against such hazardous chemicals. Additionally, the function of human macrophages is analogous to the function of plasmatocytes. Hence, this work could possibly aid us in studying the response of human immune cells against organophosphates like malathion.

Cell Surface Hsp90- and αMβ2 Integrin-Mediated Uptake of Bacterial Flagellins to Activate Inflammasomes by Human Macrophages.

All-trans retinoic acid (ATRA) is an active metabolite of vitamin A, which plays an important role in the immune function. Here, we demonstrated that ATRA induces the heat shock protein (Hsp) 90 complex on the surface of THP-1 macrophages, which facilitates the internalization of exogenous bacterial flagellins to activate the inflammasome response. Mass spectrometric protein identification and co-immunoprecipitation revealed that the Hsp90 homodimer interacts with both Hsp70 and αMβ2 integrin. ATRA-induced complex formation was dependent on the retinoic acid receptor (RAR)/retinoid X receptor (RXR) pathway and intracellular calcium level and was essential for triggering the internalization of bacterial flagellin, which was clathrin dependent. Notably, in this process, αMβ2 integrin was found to act as a carrier to deliver flagellin to the cytosol to activate the inflammasome, leading to caspase-1 activity and secretion of interleukin (IL)-1β. Our study provides new insights into the underlying molecular mechanism by which exogenous bacterial flagellins are delivered into host cells without a bacterial transport system, as well as the mechanism by which vitamin A contributes to enhancing the human macrophage function to detect and respond to bacterial infection.

Polyethylene glycol 20 kDa-induced vacuolation does not impair phagocytic function of human monocyte-derived macrophages.

Conjugation to polyethylene glycol (PEG) is commonly used to enhance drug delivery and efficacy by extending the half-life of the drug molecule. This has important implications for reducing treatment burden in diseases that require chronic prophylaxis, such as hemophilia. Clearance of PEG molecules with high molecular weights (≥ 40 kDa) has been reported to cause cellular vacuolation in mammals. Rurioctocog alfa pegol (PEGylated recombinant coagulation factor VIII) contains a 20-kDa PEG. This study investigated the effects of exposure to 20-kDa PEG (10 μg/ml to 10 mg/ml) on the morphology and function of human monocyte-derived macrophages (MDMs) in vitro. Exposure to PEG for 24 hours was associated with significant vacuolation only at concentrations of 1 mg/ml or more, which far exceed the levels associated with clinically relevant doses of rurioctocog alfa pegol. Immunofluorescence staining of PEG was detected in the cytoplasm of MDMs, indicating uptake into the cells. No impairment of MDM phagocytic activity (ability to ingest fluorescently labeled Escherichia coli) was observed with 24-hour exposure to PEG, even at concentrations associated with significant vacuolation. Furthermore, PEG exposure did not have significant effects on cytokine secretion in resting or lipopolysaccharide-stimulated MDMs, or on the expression of cell surface markers in stimulated MDMs. Cell viability was not affected by 24-hour exposure to PEG. In conclusion, vacuolation of human MDMs after exposure to 20-kDa PEG only occurred with PEG concentrations far in excess of those equivalent to clinically relevant doses of rurioctocog alfa pegol and did not affect MDM viability or functionality. Together, these results support the concept that PEG-mediated vacuolation is an adaptive cellular response rather than a toxic effect.

Resveratrol and ω-3 PUFAs Promote Human Macrophage Differentiation and Function.

Monocytes differentiate into M1 and M2 macrophages, which are classically activated by microbial products such as LPS or IFN-γ and interleukins (e.g., the anti-inflammatory and Th2 promoting IL-4), respectively. The contribution of nutrients or nutrient-based substances such as ω-3 polyunsaturated fatty acids (ω-3 PUFAs) and resveratrol (Res) on the differentiation and function of M1 and M2 macrophages was evaluated. THP-1 cells and peripheral blood mononuclear cells (PBMCs) were differentiated into M1 and M2 cells and activated with LPS/IFN-γ or IL-4/IL-13. Macrophage lineage specific surface determinants (e.g., CD11b, CD11c, CD14, CD206, CD209, CD274, HLA-DR, CCR7, CCR2) were analysed by cytofluorometry. Res and ω-3 PUFAs altered CD14, CD206, CD274 and HL-DR surface expression patterns in M1 and M2 macrophages differentiated from PBMC. LPS/IFN-γ or IL-14/IL-13 activated macrophages subpopulations, which secreted cytokines and chemokines as measured by multiplex ELISA. Res and ω-3 PUFA reduced IL-1β, IL-6, TNF-α, CXCL10/IP-10, CCL13/MCP-4 and CCL20/MIP-3α in LPS/IFN-γ activated human leukaemia THP-1 cells, which is indicative of a dampening effect on M1 macrophages. However, Res increased M1 prototypic cytokines such as IL-1β or IL-6 in macrophages derived from PBMCs and also modified the expression of IL-12p70. Collectively, Res and ω-3 PUFAs distinctly promoted the differentiation and function of M1 and M2 macrophages. We conclude that these substances strengthen the macrophage-mediated effects on the innate and adaptive immune response.

Differential immunomodulatory effects of six pesticides of different chemical classes on human monocyte-derived macrophage functions

Exposure to pesticides through eyes, skin, ingestion and inhalation may affects human health by interfering with immune cells, such as macrophages. We evaluated, in vitro, the effect of six pesticides widely used in apple arboriculture on the functions of human monocyte-derived macrophages (hMDMs). hMDMs were cultured for 4 or 24 h with or without pesticides (0.01, 0.1, 1, 10 μmol.L-1). We showed that chlorpyrifos, thiacloprid, thiophanate, boscalid, and captan had little toxic effect at the tested concentrations, while dithianon had low-cytotoxicity at 10 μmol.L−1. While boscalid showed no effect on hMDMs function, thiophanate (0.01 μmol.L−1) stimulated with TPA and thiacloprid (1, 10 μmol.L−1) stimulated with zymosan activated ROS production. Chlorpyrifos, dithianon, and captan inhibited ROS production and TNF-α, IL-1β pro-inflammatory cytokines. We established that dithianon (0.01–1 μmol.L−1) and captan (0.1, 1 μmol.L−1) induced mRNA expression of NQO1 and HMOX1 antioxidant enzymes. Dithianon also induced the mRNA expression of catalase, superoxide dismutase-2 at 10 μmol.L−1. Together, these results show that exposure to chlorpyrifos, dithianon, and captan induce immunomodulatory effects that may influence the disease fighting properties of monocytes/macrophages while pesticides such as thiacloprid, thiophanate and boscalid have little influence.