Mouse Macrophage Cell Line J774 Research Articles

Extracellular Cold Shock Protein YB-1 Induces Tolerance to GMDP and LPS in Mouse Macrophage Cell Line J774

Extracellular Cold Shock Protein YB-1 Induces Tolerance to GMDP and LPS in Mouse Macrophage Cell Line J774

Extracellular Cold Shock Protein YB-1 Induces Tolerance to GMDP and LPS in Mouse Macrophage Cell Line J774

Cold shock protein YB-1 is involved in the regulation of a huge number of fundamental biological processes. Previously, we showed that YB-1 is also involved in the process of recognition of muramylpeptide GMDP by the innate immune receptor NOD2 and is able upon preliminary administration to protect mice from death in a model of septic shock induced by Escherichia coli bacteria. We hypothesized that its protective effect may be associated with the development of a state of tolerance (“nonresponsiveness”). Changes in the cellular response were assessed by the level of mRNA expression of the target molecules by quantitative PCR analysis combined with reverse transcription. We tested the possibility of tolerance induction by the YB-1 protein in a model system on the J774 mouse macrophage cell line with the participation of E. coli bacterial cell wall components, immunostimulants GMDP (NOD2 receptor agonist) and LPS (TLR4 receptor agonist). Pretreatment of cells with YB-1 resulted in a significant decrease in the level of mRNA expression of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in response to further stimulation with GMDP and LPS, as well as significant changes in the expression of mRNA of RIP2 and MyD88 adapter molecules and components of transcriptional factor NF-κB. Our data show that YB-1 is able to induce tolerance to such as GMDP and LPS immunostimulants, apparently by increasing the production of the anti-inflammatory cytokine IL-1Ra and the SOCS1 inhibitor. A more precise characterization of the features of the YB-1-induced tolerogenic immune response requires further research.

Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages

The activation of P2X7 is a well-known stimulus for the NLRP3-caspase 1 inflammasome and subsequent rapid IL-1β secretion from monocytes and macrophages. Here we show that positive allosteric modulators of P2X7, ginsenosides, can enhance the release of three important cytokines, IL-1β, IL-6 and TNF-α from LPS-primed rodent macrophages using the J774 mouse macrophage cell line and primary rat peritoneal macrophages. We compared the immediate P2X7 responses in un-primed and LPS-primed macrophages and found no difference in calcium response amplitude or kinetics. These results suggest that under inflammatory conditions positive allosteric modulators are capable of increasing cytokine secretion at lower concentrations of ATP, thus boosting the initial pro-inflammatory signal. This may be important in the control of intracellular infections.

Macrophage colony-stimulating factor potentially induces recruitment and maturation of macrophages in recurrent pituitary neuroendocrine tumors.

Although pituitary neuroendocrine tumors (PitNETs) are usually benign, some are highly invasive and recurrent. Recurrent PitNETs are often treatment-resistant and there is currently no effective evidence-based treatment. Tumor-associated macrophages (TAMs) promote tumor growth in many cancers, but the effect of TAMs on PitNETs remains unclear. This study investigated the role of TAMs in the incidence of recurrent PitNETs. Immunohistochemical analysis revealed that the densities of CD163- and CD204-positive TAMs tended to increase in recurrent PitNETs. Compared with TAMs in primary lesions, those in recurrent lesions were enlarged. To clarify the cell-cell interactions between TAMs and PitNETs, in vitro experiments were performed using a mouse PitNET cell line AtT20 and the mouse macrophage cell line J774. Several cytokines related to macrophage chemotaxis and differentiation, such as M-CSF, were elevated significantly by stimulation with macrophage conditioned medium. When M-CSF immunohistochemistry analysis was performed using human PitNET samples, M-CSF expression increased significantly in recurrent lesions compared with primary lesions. Although no M-CSF receptor (M-CSFR) expression was observed in tumor cells of primary and recurrent PitNETs, flow cytometric analysis revealed that the mouse PitNET cell line expressed M-CSFR. Cellular proliferation in mouse PitNETs was inhibited by high concentrations of M-CSFR inhibitors, suggesting that cell-to-cell communication between PitNETs and macrophages induces M-CSF expression, which in turn enhances TAM chemotaxis and maturation in the tumor microenvironment. Blocking the M-CSFR signaling pathway might be a novel therapeutic adjuvant in treating recurrent PitNETs.

NCOA4 Regulates Iron Recycling and Responds to Hepcidin Activity and Lipopolysaccharide in Macrophages.

Macrophages, via erythrophagocytosis, recycle iron from effete erythrocytes to newly developing red blood cells. Conversion of potentially cytotoxic levels of iron from its heme into nonheme form during iron recycling is safely accomplished via coordinated regulations of cellular iron transport and homeostasis. Herein, we demonstrate the roles and regulation of NCOA4 (nuclear receptor coactivator 4)-mediated ferritinophagy in macrophages after erythrophagocytosis using the mouse macrophage cell line J774 cells. Ferritin in J774 cells increased with the rise of nonheme iron by erythrocyte ingestion and declined when total cellular iron contents subsequently decreased. NCOA4, a selective autophagic cargo receptor for ferritin, was responsible for the control of cellular ferritin and total iron contents at the later stage of erythrophagocytosis. A hepcidin analog, which limits the flux of iron through iron-recycling by inhibiting iron export at the plasma membrane, repressed NCOA4 expression and led to accumulation of ferritin in the mouse macrophages. Transcriptome analyses revealed a functional association of immune response with NCOA4-dependent gene expressions, and we confirmed repression of Ncoa4 by lipopolysaccharide (LPS) in J774 cells and the spleen of mice. Collectively, our studies indicate that NCOA4 facilitates cellular ferritin turnover and the release of iron by macrophages after erythrophagocytosis and functions as a regulatory target for molecular signals of systemic iron overload and inflammation. These identify macrophage NCOA4 as a potential therapeutic target for disorders of systemic iron dysregulation, including anemia of inflammation and hemochromatosis.

Effects of pre-eclampsia on HDL-mediated cholesterol efflux capacity after pregnancy

Background and aimsPreeclampsia (PE) is associated with life-long increased risk of cardiovascular disease. One of the main protective functions of high-density lipoprotein (HDL) is its role in reverse cholesterol transport. HDL-mediated cholesterol efflux capacity (CEC) is decreased during pregnancy in women with PE. Whether this persists postpartum is unknown. MethodsBasal and transporter-specific CEC were determined 6 months postpartum in women who had a normotensive (n = 44) or a PE (n = 42) pregnancy. CEC was also measured in 23 normotensive and 20 PE women for whom samples were collected 24 months postpartum. Basal, ATP-binding cassette transporter-A1 (ABCA1)- and -G1 (ABCG1)-specific CEC were primarily determined using Chinese hamster ovary cells stably expressing human ABCA1 or ABCG1, and were also assessed using a J774 mouse macrophage cell line. ResultsABCA1-specific CEC was significantly lower in women who had PE 6 months postpartum (0.57 ± 0.1 vs 0.53 ± 0.08; p < 0.05), whilst basal and ABCG1-specific efflux were not significantly different. cAMP-specific CEC in J774 cells was also lower 6 months after PE (0.85 ± 0.21 vs 0.75 ± 0.25, p < 0.05). Although apoA-I, apoE, plasminogen and PON-1 levels were not significantly different in women who had PE compared with controls, ABCA1 efflux did correlate with apoA-l, HDL-C and apoE levels after a normal, and with apoA-l and HDL-C levels after a PE pregnancy. ABCA1-specific efflux decreased in all women between 6 and 24 months postpartum, by 11 ± 1.6% in women who had a normotensive pregnancy and 9 ± 1.3% in women who had PE. After adjustment for apoA-I levels, there was no significant difference in ABCA1-specific efflux between the groups at 6 months postpartum and in normotensive women over time, but remained significantly different between 6 and 24 months in women who had PE. ConclusionsABCA1-mediated CEC is impaired 6 months postpartum after a PE pregnancy and decreases thereafter in both normotensive and PE pregnancies. ABCA1-mediated efflux is dynamic after pregnancy but is unlikely to explain the long-term increased CVD risk in women with PE.

Trichoderma stromaticum spores induce autophagy and downregulate inflammatory mediators in human peripheral blood-derived macrophages.

Trichoderma spp. are usually considered safe and normally used as biocontrol and biofertilization. Safety for human health is evaluated by several tests that detect various effects such as allergenicity, toxicity, infectivity, and pathogenicity. However, they do not evaluate the effects of the agent upon the immune system. The aim of this study was to investigate the interaction between T. stromaticum spores and mammalian cells to assess the immunomodulatory potential of the spores of this fungus. First, mouse macrophage cell line J774 and human macrophages were exposed to fungal spores and analyzed for structural features, through scanning and transmission electron microscopy. Then, various analysis were performed in human macrophages as to their effect in some functional and molecular aspects of the immune system through immunocytochemistry, flow cytometry and gene expression assays. We demonstrated that T. stromaticum spores induces autophagy and autophagy-related genes (ATGs) and downmodulate inflammatory mediators, including ROS, NLRP3, the cytokines IL-1β, IL-18, IL-12 and IL-10, as well as TLR2, TLR4, miR-146b and miR-155, which may lead to an augmented susceptibility to pathogens. Our study shows the extension of damages the biofungicide Tricovab® can cause in the innate immune response. Further studies are necessary to elucidate other innate and adaptive immune responses and, consequently, the safety of this fungus when in contact with humans.

Total RNA and genomic DNA of Lactobacillus gasseri OLL2809 induce interleukin-12 production in the mouse macrophage cell line J774.1 via toll-like receptors 7 and 9

BackgroundLactobacillus gasseri OLL2809 can highly induce interleukin (IL)-12 production in immune cells. Even though beneficial properties of this strain for both humans and animals have been reported, the mechanism by which the bacteria induces the production of IL-12 in immune cells remains elusive. In this study, we investigated the mechanism of induction of IL-12 using a mouse macrophage cell line J774.1.ResultsInhibition of phagocytosis of L. gasseri OLL2809, and myeloid differentiation factor 88 and Toll-like receptors (TLRs) 7 and 9 signalling attenuated IL-12 production in J774.1 cells. Total RNA and genomic DNA of L. gasseri OLL2809, when transferred to the J774.1 cells, also induced IL-12 production. The difference in the IL-12-inducing activity of Lactobacilli is attributed to the susceptibility to phagocytosis, but not to a difference in the total RNA and genomic DNA of each strain.ConclusionWe concluded that total RNA and genomic DNA of phagocytosed L. gasseri OLL2809 induce IL-12 production in J774.1 cell via TLRs 7 and 9, and the high IL-12-inducing activity of L. gasseri OLL2809 is due to its greater susceptibility to phagocytosis.

AnIn VitroInvestigation of the Effects of the Glucocorticoid on Leishmania major Amastigotes

Background: Leishmaniasis is a vector-borne disease caused by Leishmania species. The transcription factor (NF-κB) activates and the innate immune system starts working when this parasite attacks macrophages. In addition, glucocorticoids increase nitric oxide (NO) and INFγ leads to the apoptosis of the cell by inhibiting the NF-κB activity. The aim of this study was the in vitro investigation of the effects of the glucocorticoids on Leishmania major amastigotes. Methods: Leishmania major was produced in a massive volume. Then, promastigotes penetrated into macrophages and converted to amastigotes by adding promastigotes to the J774 mouse macrophage cell line and providing appropriate conditions. Next, the infected macrophages with L. major parasite were treated with different concentrations of prednisolone and mometasone. After 24 and 48 hours, the effect of the drugs was evaluated based on the average number of amastigotes in the infected macrophages. In addition, the amount of NO and the mean interleukin 12 (IL-12) level secreted by the infected macrophages treated with different concentrations of drugs were measured by Griess reagent and ELISA reader, respectively. Results: The average number of amastigotes in the infected macrophages treated with different concentrations of the drug was significantly different from the control group. Further, the amount of NO and the mean level of IL-12 secreted by infected macrophages had a direct and significant relationship with different concentrations of drugs, but the results of Tukey post hoc test showed that the reduction in the number of promastigotes was not time-dependent. Conclusions: In general, prednisolone and mometasone stimulated macrophages increased the IL-12 levels and NO secretion and finally decreased the number of parasites in infected macrophages.

Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-carbohydrazides derivatives as modifiers of redox homeostasis of Trypanosoma cruzi

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC50=5µM and 7.5µM, respectively) and less than nifurtimox (IC50=3.6µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC50 values of 160.64 and 160.66µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively.

RNA and a cell wall component of Enterococcus faecalis IC-1 are required for phagocytosis and interleukin 12 production by the mouse macrophage cell line J774.1

Enterococcus faecalis is a resident lactic acid bacterium in the human intestine. Its immunostimulatory action was reported to be enhanced by heat sterilization. To investigate its beneficial actions, we evaluated the ability of 10 E. faecalis strains to induce interleukin-12 (IL-12) production in a mouse macrophage cell line, J774.1 and found that the strain, E. faecalis IC-1, had a potent IL-12-inducing ability. Furthermore, we investigated the underlying mechanism by treating IC-1 cells with RNase or lysozyme. Its activity almost disappeared and an antagonist of Toll-like receptor (TLR) 7 inhibited this activity. Moreover, lysozyme-treated IC-1 bacteria were not phagocytized by J774.1 cells, and did not induce IL-12 production. Based on our results, we propose that macrophages recognize the cell wall components of IC-1, leading to phagocytosis. The IC-1 RNA is then recognized by TLR7, which induces the production of IL-12.

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains.

Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5'end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli The recombinant fusion hepcidin-ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin-ferroportin interaction in cells and also as drug-delivery agent.

Mycobacterium avium subsp. paratuberculosis down-regulates mRNA expression of iron-induced macrophage Ferroportin 1

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease. The mechanisms by which MAP is able to adapt to the innate host response are still unclear. We examined Ferroportin 1 (FPN1) mRNA expression levels via real-time PCR of the mouse macrophage cell line J774 that was incubated in the presence of Mycobacterium avium subsp. paratuberculosis (MAP) or MAP crude protein extract. Infection with live MAP decreased FPN1 mRNA levels in a multiplicity of infection (MOI)-dependent fashion. Macrophages infected with MOIs of 20:1 and 15:1 did not show any change in FPN1 gene expression, whereas MOIs of 10:1 and 5:1 induced a decrease of 50 and 80%, respectively. Macrophages treated with 50, 100, 150 and 200 µg/mL of MAP crude extract (ATCC19698) decreased FPN1 mRNA expression by 25%. Additionally, up-regulation of FPN1 mRNA by an iron overload treatment of 400 µM of ferric nitrilotriacetate (FeNTA) was abrogated by live MAP (MOI 20:1) by approximately 70%. Our data revealed an inhibitory effect of MAP on FPN1 mRNA and suggested a bacterial mechanism that may play a role in host iron regulation.

Mycobacterium avium subsp. paratuberculosis down-regulates mRNA expression of iron-induced macrophage Ferroportin 1

Veterinaria México OA ISSN: 2448-6760Cite this as:Landeros Sánchez B, Gutierrez Pabello JA, Medina Basulto GE, Renteria Evangelista TB, Díaz Aparicio E, Oshima S. Mycobacterium avium subsp. paratuberculosis down-regulates mRNA expression of iron-induced macrophage Ferroportin 1. Veterinaria México OA. 2016;3(1). doi: 10.21753/vmoa.3.1.361Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease. The mechanisms by which MAP is able to adapt to the innate host response are still unclear. We examined Ferroportin 1 (FPN1) mRNA expression levels via real-time PCR of the mouse macrophage cell line J774 that was incubated in the presence of Mycobacterium avium subsp. paratuberculosis (MAP) or MAP crude protein extract. Infection with live MAP decreased FPN1 mRNA levels in a multiplicity of infection (MOI)-dependent fashion. Macrophages infected with MOIs of 20:1 and 15:1 did not show any change in FPN1 gene expression, whereas MOIs of 10:1 and 5:1 induced a decrease of 50 and 80%, respectively. Macrophages treated with 50, 100, 150 and 200 µg/mL of MAP crude extract (ATCC19698) decreased FPN1 mRNA expression by 25%. Additionally, up-regulation of FPN1 mRNA by an iron overload treatment of 400 µM of ferric nitrilotriacetate (FeNTA) was abrogated by live MAP (MOI 20:1) by approximately 70%. Our data revealed an inhibitory effect of MAP on FPN1 mRNA and suggested a bacterial mechanism that may play a role in host iron regulation.Figure 1. Infection by Mycobacterium avium subsp. paratuberculosis (MAP) down-regulates macrophage fpn1 gene expression. Murine macrophages, J774, were infected with MAP at different multiplicities of infection (MOIs) for 6 hours. Total RNA was extracted and gene expression was measured by real-time PCR. Results were normalized to GAPDH and expressed as units of relative expression (URE). Results are the mean ± standard deviation of 3 independent experiments with 3 replicates each. Statistical significance, P = 0.05.

Proinflammatory effect of AbetaPP induced ST6GAL1 secretion from C2C12 myogenic cell line

Objective: ST6 beta-galactosamide α-2,6-sialyltranferase 1 (ST6GAL1), the major α2,6- sialyltransferase responsible for the broad synthesis of glycoproteins and glycolipids, is another physiological substrate of Beta site APP-cleaving enzyme 1 (BACE1) other than amyloidbeta precursor protein (AbetaPP). We have previously shown that AbetaPP overexpression in C2C12 mouse myoblast cell line increased the expression and secretion of ST6GAL1 enzyme under in vitro conditions. Since the secretion of ST6GAL1 is known to be enhanced during inflammation, we investigated whether AbetaPP induced ST6GAL1 secretion from C2C12 cells affected proinflammatory cytokine production by J774 mouse macrophage cell line.

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis.

Sensitization of murine macrophages and human hepatoma cells to lipopolysaccharide-induced oxidative and nitrosative stress by aspirin

Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is one of the most commonly used anti-inflammatory drugs. Our aim was to study the mechanism of aspirin action in the presence of LPS in murine macrophages and human hepatoma cells. We investigated the effects of LPS with a subtoxic concentration of ASA, alone and in conjunction, on oxidative and nitrosative stress using the mouse macrophage cell line J774.2 and human hepatoma HepG2 cells. Apoptosis was measured by flow cytometric analysis. LPS alone markedly induced nitric oxide (NO) production in macrophage cells and moderately in HepG2 cells. When ASA was added to LPS-treated macrophage cells, the increase in NO production was significantly higher than that induced by LPS or ASA alone. However, treatment of HepG2 cells with LPS or ASA alone or in combination exhibited moderate effects on NO production. Similarly, production of ROS after treatment with LPS and ASA was also higher in macrophages than in HepG2 cells. The combination of LPS and ASA also markedly increased the rate of lipid peroxidation in both cell lines. The activity of aconitase, an oxidative stress-sensitive mitochondrial matrix enzyme, was also markedly inhibited in J774.2 cells compared with HepG2 cells. Furthermore, treatment of cells with LPS alone or in combination with ASA also resulted in higher apoptotic cell death in macrophages. These results suggest that macrophages are more sensitive to LPS and that LPS and ASA treatments synergistically increase oxidative and nitrosative stress in these cells.

Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase‐1

3',5'-Cyclic nucleotide PDE4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5'AMP, down-regulating cAMP signalling in cells. MAPK phosphatase-1 (MKP-1) is an endogenous p38 MAPK signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP-1 expression and whether MKP-1 is involved in the anti-inflammatory effects of rolipram. The effect of rolipram on TNF production was investigated in J774 mouse macrophage cell line and in primary mouse peritoneal macrophages (PM) from wild-type (WT) and MKP-1(-/-) mice. We also investigated the effect of rolipram on carrageenan-induced paw inflammation in WT and MKP-1(-/-) mice. MKP-1 expression was enhanced by rolipram, by a non-selective PDE inhibitor IBMX and by a cAMP analogue 8-Br-cAMP in J774 cells and in PM. Enhanced MKP-1 mRNA expression by rolipram was reversed by a PKA inhibitor. Rolipram, IBMX and 8-Br-cAMP also inhibited TNF production in activated macrophages. Accordingly, rolipram inhibited TNF production in PMs from WT mice but, interestingly, not in PMs from MKP-1(-/-) mice. Furthermore, rolipram attenuated carrageenan-induced paw inflammation in WT but not in MKP-1(-/-) mice. PDE4 inhibitor rolipram was found to enhance the expression of MKP-1, and MKP-1 mediated, at least partly, the anti-inflammatory effects of PDE4 inhibition. The results suggest that compounds that enhance MKP-1 expression and/or MKP-1 activity hold potential as novel anti-inflammatory drugs.

Immunomodulatory Impact of Leishmania-Induced Macrophage Exosomes: A Comparative Proteomic and Functional Analysis

Released by many eukaryotic cells, the exosomes are 40–100 nm vesicles shown to operate over the complex processes of cell-cell communication. Among the metazoan cell lineages known to generate exosomes is the mononuclear phagocyte lineage, a lineage that parasites such as Leishmania are known to subvert as host cells. We previously reported that mouse macrophage signaling and functions are modified once co-incubated with exoproteome of Leishmania promastigotes. Using mass spectrometry analysis, we were curious to further compare the content of purified exosomes released by the J774 mouse macrophage cell line exposed or not to either LPS or to stationary phase Leishmania mexicana promastigotes. Collectively, our analyses resulted in detection of 248 proteins, ∼50–80% of which were shared among the three sources studied. Using exponentially modified protein abundance index (emPAI) and network analyses, we found that the macrophage exosomes display unique signatures with respect to composition and abundance of many functional groups of proteins, such as plasma membrane-associated proteins, chaperones and metabolic enzymes. Moreover, for the first time, L. mexicana surface protease GP63 is shown to be present in exosomes released from J774 macrophages exposed to stationary phase promastigotes. We observed that macrophage exosomes are able to induce signaling molecules and transcription factors in naive macrophages. Finally, using qRT-PCR, we monitored modulation of expression of multiple immune-related genes within macrophages exposed to exosomes. We found all three groups of exosomes to induce expression of immune-related genes, the ones collected from macrophages exposed to L. mexicana sharing properties with exosomes collected from macrophage left unexposed to any agonist. Overall, our results allowed depicting that protein sorting into macrophage-derived exosomes depends upon the cell status and how such distinct protein sorting can in turn impact the functions of naive J774 cells.

ChemInform Abstract: Regio‐ and Stereoselective C10β—H Functionalization of Sinomenine: An Access to More Potent Immunomodulating Derivatives.

AbstractAll newly synthesized compounds are evaluated for their pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) for the mouse macrophage cell line J774.