The inflammatory effects of a repeated exposure of human macrophages to PM2.5 from house dust-SRM 2585 are partially reversible and disrupt the LPS/CD14 signaling pathways.

Inhalation of crystalline silica (SiO2) is a risk factor of systemic autoimmune diseases such as systemic sclerosis (SSc) and fibrotic pulmonary disorders such as silicosis. A defect of apoptotic cell clearance (i.e., efferocytosis, a key process in the resolution of inflammation) is reported in macrophages from patients with fibrotic or autoimmune diseases. However, the precise links between SiO2 exposure and efferocytosis impairment remain to be determined. Answering to this question may help to better link innate immunity and fibrosis. In this study, we first aim to determine whether SiO2 might alter efferocytosis capacities of human and mouse macrophages. We secondly explore possible mechanisms explaining efferocytosis impairment, with a specific focus on macrophage polarization and on the RhoA/ROCK pathway, a key regulator of cytoskeleton remodeling and phagocytosis. Human monocyte-derived macrophages (MDM) and C57BL/6J mice exposed to SiO2 and to CFSE-positive apoptotic Jurkat cells were analyzed by flow cytometry to determine their efferocytosis index (EI). The effects of ROCK inhibitors (Y27632 and Fasudil) on EI of SiO2-exposed MDM and MDM from SSc patients were evaluated in vitro. Our results demonstrated that SiO2 significantly decreased EI of human MDM in vitro and mouse alveolar macrophages in vivo. In human MDM, this SiO2-associated impairment of efferocytosis, required the expression of the membrane receptor SR-B1 and was associated with a decreased expression of M2 polarization markers (CD206, CD204, and CD163). F-actin staining, RhoA activation and impairment of efferocytosis, all induced by SiO2, were reversed by ROCK inhibitors. Moreover, the EI of MDM from SSc patients was similar to the EI of in vitro- SiO2-exposed MDM and Y27632 significantly increased SSc MDM efferocytosis capacities, suggesting a likewise activation of the RhoA/ROCK pathway in SSc. Altogether, our results demonstrate that SiO2 exposure may contribute to the impairment of efferocytosis capacities of mouse and human macrophages but also of MDM in SiO2-associated autoimmune diseases and fibrotic disorders such as SSc; in this context, the silica/RhoA/ROCK pathway may constitute a relevant therapeutic target.

Liver X receptors (LXRs) are ligand-activated transcription factors involved in the control of lipid metabolism and inflammation. Several studies have recently shown that LXRs promote reverse cholesterol transport and inhibit atherosclerosis. Our study investigated whether LXRs affect macrophage uptake of LDL by human monocyte-derived macrophages. We have previously shown that human monocytes differentiated into macrophages with macrophage-colony-stimulating factor (M-CSF) constitutively take up large amounts of native LDL by receptor-independent, fluid-phase pinocytosis. In the research reported here, human monocytes were differentiated to macrophages in the presence of M-CSF with or without the LXR agonists T0901317 or 22(R)-hydroxycholesterol. Then, macrophages were incubated with native (125)I-LDL to determine LDL uptake. T0901317 and 22(R)-hydroxycholesterol inhibited (125)I-LDL uptake by 68 +/- 1% and 69 +/- 2%, respectively, and decreased pinocytotic vacuoles in the macrophages. (125)I-BSA uptake, a measure of fluid-phase pinocytosis, and (125)I-LDL uptake were the same, and T0901317 treatment inhibited uptake of both to the same degree. T0901317 did not affect receptor-mediated uptake of acetylated LDL, showing that the LXR effect is specific for fluid-phase pinocytosis of lipoproteins. Our results show that LXRs downregulate macrophage pinocytosis of LDL. The findings reveal an additional new mechanism by which LXR agonists may inhibit macrophage cholesterol accumulation and atherosclerosis, namely, by inhibiting macrophage uptake of LDL.

Alamandine, a protective component of the renin-angiotensin system, reduces cellular proliferation and interleukin-6 secretion in human macrophages through MasR–MrgDR heteromerization

Nox4 is member of the family of NADPH oxidase expressed in endothelium and smooth muscle cells, but has not previously been reported in human monocytes or monocyte-derived macrophages. We now identified Nox4 as a new Nox member in human monocytes and monocyte-derived macrophages. Nox4 and the phagocytic NADPH oxidase gp91/Nox2 were differentially regulated during monocyte differentiation. In contrast to Nox2, Nox4 protein expression was upregulated in mature human macrophages, and confocal microscopy revealed that Nox4 colocalized primarily with mitochondria. Exposure of macrophages to OxLDL for 24 h further increased Nox4 expression in a concentration-dependent manner. OxLDL-induced Nox4 expression was blocked by inhibitors of MEK1/2, UO126 and PD98059, but not by SB203580 and SP600125, inhibitors of p38MAPK or JNK, respectively. Furthermore, MEK inhibitors, but not inhibitors of p38MAPK or JNK, protected macrophages from OxLDL-induced cell injury, identifying a MEK/ERK1/2-dependent signaling pathway upstream of OxLDL-induced Nox4 expression that is involved in macrophage death. Previously, we showed that OxLDL stimulates the intracellular formation of reactive oxygen species (ROS) in human macrophages, but the source of these ROS remained elusive. We also demonstrated that the peroxyl radical scavenger Trolox protects macrophages from OxLDL-induced cell injury. Interestingly, Trolox did not prevent Nox4 expression induced by OxLDL, suggesting that Trolox acts downstream of OxLDL-induced Nox4 expression. These results not only identify Nox4 as a new intracellular source of ROS in macrophages, but also implicate Nox4 in OxLDL-induced ROS formation and macrophage death, which may have important implications for the pathogenesis of atherosclerosis.

Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C.) burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM) represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III) to high (MLVA group IV). Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80) on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups.

We have shown recently that lipoprotein-proteoglycan complexes isolated from human atherosclerotic lesions stimulated cholesteryl ester synthesis in human monocyte-derived macrophages [Vijayagopal, Srinivasan, Radhakrishnamurthy and Berenson (1992) Arterioscler. Thromb. 12, 237-249]. The present study was conducted to determine the mechanism of cellular uptake of the complexes. A chondroitin sulphate-dermatan sulphate proteoglycan was isolated from normal human aorta and complexed to 125I-labelled human low-density lipoprotein (LDL). The binding and degradation of 125I-LDL-proteoglycan complex were then studied in human monocyte-derived macrophages. The specific binding and degradation of the complex showed saturability and concentration-dependency. The Kd for binding was 1.5 x 10(-8) M, which was greater than that reported for LDL in monocyte-derived macrophages. Binding of the complex was not subject to down-regulation. Chloroquine inhibited degradation of the complex and the resultant stimulation of cholesteryl ester synthesis. Limited treatment of macrophages with proteolytic enzymes abolished binding and degradation of the complex significantly. Macrophages bound 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. Excess LDL and proteoglycan did not compete against the binding of the complex; however, excess acetyl-LDL competed for 61% of the binding. Likewise, excess LDL-proteoglycan complex inhibited the binding of 125I-acetyl-LDL by 64%. Polyinosinic acid and cytochalasin D inhibited the binding of 125I-LDL-proteoglycan complex by 60% and 36% respectively. Compared with that of acetyl-LDL, the degradation of LDL-proteoglycan complex was retarded in human macrophages. The results indicate that the uptake of LDL-proteoglycan complex in human monocyte-derived macrophages is not mediated through binding to the LDL receptor; but occurs predominantly via the scavenger receptor, with phagocytosis playing a minor role in the process.

<p>The Leishmania spp. are kinetoplastid protozoan parasites that cause a spectrum of highly prevalent and neglected tropical diseases known as leishmaniasis. The parasites must undergo two life forms during their life cycle: the extracellular promastigote life stage within the sand fly vector, and the intracellular amastigote life stage after internalization of host phagocytic cells. In the extracellular life stage, Leishmania promastigotes reside and develop to their infectious metacyclic form solely in the gut lumen of the sand fly, a process known as metacyclogenesis. During this process, other organisms that co-inhabit the sand fly gut, collectively known as the microbiome, influence parasite development. Based on the hypothesis that vector gut microbiota influence the development of parasite virulence, we sequenced midgut microbiomes of the sand fly Lutzomyia longipalpis with or without L. infantum infection. Sucrose fed sand flies contained a highly diverse, stable midgut microbiome. Blood feeding caused a decrease in bacterial richness, which eventually recovered. However, bacterial richness progressively decreased in L. infantum-infected sand flies. Furthermore, parasites altered the relative abundance of several bacterial phylogenies, including Pseudomonas and Serratia. Importantly, antibiotic-mediated perturbation of the midgut microbiome rendered sand flies unable to support parasite growth and consequent development to infectious metacyclic forms, and revealing the level of microbial diversity may induce flies resistant to infection. Together, these data suggest the sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation prior to disease transmission.</p> <p>During the intracellular amastigote life form, macrophages are the primary cell type to phagocytize parasites. The effect of secreted factors such as exosomes from Leishmania-infected human cells and their effect on the immune response has not been extensively investigated. In this thesis, we characterized the proteome of primary human donor monocyte-derived macrophage (MDM) exosomes during L. infantum infection compared to donor-matched uninfected controls, and determined their impact on naïve MDMs measured by cytokine gene expression and resistance to subsequent parasite infection. Proteomic comparisons of infected and uninfected MDM exosomes were made using stable isotopic dimethyl labeling LC-MS/MS technology. A total of 484 human proteins were identified between four donors. Proteins significantly less abundant in exosomes derived from infected MDMs were matrix metalloprotease 9, galectin-3 binding protein, and several Annexins and histone proteins. Proteins more abundant included galectin-1, galectin-9, and serotransferrin and transferrin receptor 1. Interestingly, class I and class II MHC protein chains were differentially abundant in our samples. Furthermore, we observed several Leishmania spp. proteins in exosomes from infected MDMs as well. Naïve MDMs pretreated with exosomes from infected or uninfected MDM for 4 hours were not more resistant to L. infantum infection nor displayed increased gene expression of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 or TNF-α. To date, the work presented in this thesis is the first to comprehensively identify the proteome in primary human MDM exosomes during Leishmania spp. infection, and to determine the impact of these exosomes on the immune response of other naïve human MDMs.</p>

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Cholesteryl ester-laden macrophages are the hallmark of the fatty streaks that precede arteriosclerotic plaques in humans and experimental animals. This article studies several aspects of cytoplasmic cholesteryl ester metabolism in cultured human monocyte-derived macrophages. Adenosine 3',5'-cyclic monophosphate (cAMP) consistently inhibited cholesteryl ester mobilization from cells that had been loaded with cholesteryl esters by preincubation with acetylated low-density lipoprotein. This effect was observed in both the absence and presence of extracellular cholesterol acceptors as well as with acyl coenzyme A: cholesterol acyltransferase inhibitors. In contrast, dibutyryl cAMP activated cholesteryl ester hydrolysis in J774 macrophages. Since hormone-sensitive lipase is thought to be responsible for the neutral cholesteryl ester hydrolytic activity in several cell types, we looked for the presence of its mRNA in our macrophages by means of reverse transcription coupled to the polymerase chain reaction technique. Hormone-sensitive lipase mRNA was detected in J774 macrophages but not in human monocytes or in human monocyte-derived macrophages. These results demonstrated great differences in cholesteryl ester metabolism between macrophages of different origin. While hormone-sensitive lipase may be responsible for neutral cholesteryl ester hydrolytic activity in J774 macrophages, in human monocyte-derived macrophages it is not; thus, a different and as yet unidentified enzyme must be present.

Macrophages are important sites of bacterial replication and host immune responses during Mycobacterium tuberculosis (Mtb) infection with distinct roles for alveolar macrophages (AMs) early in infection and monocyte-derived macrophages (MDMs) later in disease. Here, we leverage data from human and mouse models to perform a cross-species analysis of macrophage responses to Mtb. Overall, we find that both subsets of human and murine macrophages mount a strong interferon response to Mtb infection. However, AMs across both species do not generate as strong a pro-inflammatory response as human MDMs or murine bone marrow-derived macrophages (BMDMs), as characterized by TNFA signaling and inflammatory response pathways. Interestingly, AMs from mice that were previously vaccinated with BCG (scBCG) or from a model of contained TB (coMtb) had more similar responses to human AMs than control mice. We also identify species-specific pathways altered by infection differently in mouse and human macrophages, including cholesterol homeostasis. Lastly, to investigate downstream effects of the macrophage interferon responses, we examine expression of interleukin (IL)-10, an immunosuppressive cytokine induced by Type I Interferons, and c-Maf, a transcription factor required for myeloid IL-10 expression. We find that c-Maf and IL-10 have significantly lower expression in AMs compared to MDMs in both humans and mice, suggesting one possible mechanism by which AMs mount a stronger interferon response following Mtb infection. Overall, these results highlight the dynamics of innate myeloid responses throughout Mtb infection and the benefit of a combined analysis across species to reveal conserved and unique responses.

Endothelin-1 Synthesis and Endothelin B Receptor Expression in Human Coronary Artery Smooth Muscle Cells and Monocyte-derived Macrophages is Up-regulated by Low Density Lipoproteins

Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes

Corneal tissue induces transcription of metallothioneins in monocyte-derived human macrophages

The induction of cyclooxygenase-2 (COX-2) and the production of PGE(2) in response to pathogen-associated molecular patterns decorated with mannose moieties were studied in human monocytes and monocyte-derived macrophages (MDM). Saccharomyces cerevisiae mannan was a robust agonist, suggesting the involvement of the mannose receptor (MR). MR expression increased along the macrophage differentiation route, as judged from both its surface display assessed by flow cytometry and the ability of MDM to ingest mannosylated BSA. Treatment with mannose-BSA, a weak agonist of the MR containing a lower ratio of attached sugar compared with pure polysaccharides, before the addition of mannan inhibited COX-2 expression, whereas this was not observed when agonists other than mannan and zymosan were used. HeLa cells, which were found to express MR mRNA, showed a significant induction of COX-2 expression upon mannan challenge. Conversely, mannan did not induce COX-2 expression in HEK293 cells, which express the mRNA encoding Endo180, a parent receptor pertaining to the MR family, but not the MR itself. These data indicate that mannan is a strong inducer of COX-2 expression in human MDM, most likely by acting through the MR route. Because COX-2 products can be both proinflammatory and immunomodulatory, these results disclose a signaling route triggered by mannose-decorated pathogen-associated molecular patterns, which can be involved in both the response to pathogens and the maintenance of homeostasis.

ObjectiveActivated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs).ResultsComponents released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell –induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.ConclusionsMast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.