Murine Macrophage Cell Line J774 Research Articles

In Vitro Determination of Antileshmanial Activities of Benzimidazolium Derivatives on L. major Promastigotes and Amastigotes.

Leishmaniasis is a serious public health problem infecting millions of people worldwide. An effective and reliable treatment method to be used in the treatment of the disease has not been developed yet. In this article, the anti-leishmanial activities of two benzimidazolium derivatives (B.A and B.B) against Leishmania major promastigotes and amastigotes, which are known to cause cutaneous leishmaniasis, were investigated for the first time. The immunostimulatory activity of the developed formulations was determined using the J774 murine macrophage cell line. B.A and B.B compounds were found to have a much higher cytotoxic effect than Amphotericin B (IC50 value 0.75μM ± 0.03), which is used as the reference drug. The IC50 value was determined as 2.02µM ± 0.52 for B.A and 1.83µM ± 0.71 for B.B in Leishmania promastigotes. In addition, IC50 values of B. A and B.B Leishmania amastigotes were found to be 1.01µM and 0.67µM, respectively. It was found that B.B was 81.12 times more selective than Amphotericin B and showed the highest selectivity against L. major promastigotes (359.09) and amastigotes (980.80). Considering the selectivity indices (SI) of B.A and B.B, both compounds tested are more promising than Amphotericin B. The results showed that benzimidazolium derivatives have anti-leishmanial potential against L. major, which is the causative agent of cutaneous leishmaniasis. Thus, we can say that the obtained results will help the development of effective and safe antileishmanial drug formulations against cutaneous leishmaniasis.

P349 Relationship between genotypes and in vitro pathogenic behavior of Uruguayan strains of Cryptococcus spp.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectiveContribute to the knowledge of the relationship between genetic and pathogenic characteristics of meningeal cryptococcosis-producing strains from Uruguay.MethodsWe have included a total of 21 isolates of Cryptococcus spp from human cases of meningoencephalitis. We start from the general strain collection of the mycology laboratory of the department of parasitology and mycology, medical school, University of the Republic. The strain was sectioned taking into account the phenotypic diversity and genetic diversity through a previous analysis by PCR fingerprinting, for study by MLST.MLST was performed according to the protocol available at: http://mlst.mycologylab.org/defaultinfo.aspx?Page=CryptoHome.Infection, intracellular survival, and cell proliferation assays were performed. The murine macrophage cell line J774 (ATCC TIB 67) was used.ResultsOf the 21 strains studied, in 13 we obtained an ST that had been previously reported (9 C. neoformans complex and 4 C. gattii complex). For the remaining 8 strains, we obtained new allelic combinations to which new STs were assigned, which were deposited in the MLST database for Cryptococcus spp. (http://mlst.mycologylab.org) (Table 1).All strains were phagocytosed and all were able to persist within macrophages at 24 h post-infection, although there was great variability between strains (Table 1). In most of the strains, the control of macrophages prevailed through cell lysis mechanisms over Cryptococcus spp, with one proliferation index (PI) ˂1.0. In the proliferation assay, only 4 strains managed to proliferate within the macrophages, 3 of them correspond to C. gattii complex, genotypes VGI and VGII. The 4th strain that achieved proliferation was corresponding to the VNI genotype.ConclusionWe obtained a clear differentiation of in vitro behavior between the strains corresponding to C. neoformans complex and C. gattii complex. The strains belonging to C. gattii complex presented less phagocytosis and greater persistence and intracellular proliferation.

GRIM‐19 is a target of mycobacterial Zn 2+ metalloprotease 1 and indispensable for NLRP3 inflammasome activation

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1β production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1β production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1β production during mycobacterial infection.

Expanding the chemical space of aryloxy-naphthoquinones as potential anti-Chagasic agents: synthesis and trypanosomicidal activity

In continuation our effort to research the chemical space of aryloxy-naphthoquinones as potential anti-Chagas agents, we synthesized nine derivatives and these compounds were evaluated in vitro against the epimastigote and trypomastigote forms of Mexican strains of Trypanosoma cruzi (T. cruzi). Most of these derivatives are highly active against epimastigote forms (IC50 < 1.0 µM) compared to the reference drug benznidazole (Bzn). Then these were evaluated on trypomastigotes, which is showing better potency results than Bzn for compounds 3b and 3g. In addition, the cytotoxicity of these compounds was determined on the murine macrophage cell line J774. 3b and 3i were the most selective compounds against NINOA trypomastigote and INC-5 epimastigote forms, respectively. Further these compounds also have good oral bioavailability according to theoretical predictions. Finally, we were able to determine optimal substitution patterns using pharmacophoric models. All these results are provided very useful structural information to continue our designing of naphthoquinone derivatives against T. cruzi.

The novel piperazino-enaminone JOAB-40 reduced colitis severity in mice via inhibition tumor necrosis factor-α and interleukin-1β

Brief introductionThe synthetic compound enaminone E121 has an established role as a potent anti-tussive, bronchodilator and anti-inflammatory agent in asthma, cough, and colitis induced animal models. The addition of an N-alkylated piperazine motif to the terminal end of E121 lead to the generation of various analogues such as JOAB-40. JOAB-40 was shown to be more potent than the lead compound E121 in inhibiting the expression of the chemokine receptor CCR2, ERK1/2 phosphorylation, and the release of pro-inflammatory cytokines in vitro. Main objective of the studyWe hypothesize that JOAB-40 is more potent than the lead compound E121 in reducing colitis severity in mice in part through inhibiting the release of TNFα and IL-1β. MethodsColitis was induced by dextran sulfate sodium (DSS) administration using prophylactic and treatment approaches. The severity of the inflammation was determined by the gross (macroscopic) and histological (microscopic) assessments. The levels of TNFα, IL-1β, and IL-10 release in response to lipopolysaccharide (LPS) stimulation from the adherent murine macrophage cell line J774.2 in vitro, and the circulating levels of TNFα in vivo was measured by ELISA-based technique. Significant findings from the studyE121 administration (1–60 mg/kg) in mice with established colitis (treatment approach) did not reduce colitis severity. On the other hand, JOAB-40 administration significantly reduced colitis severity in mice when administered using two approaches; a) prophylactic (given along colitis induction), and b) treatment (given after colitis was established) with doses as low as 10 mg/kg. The degree of inhibition of TNFα and IL-1β (but not IL-10) release from J774.2 cell line in response to LPS stimulation was more potent with JOAB-40 than E121. This was also observed in vivo in regards to the circulating levels of TNFα. Relevant contribution to knowledgeOur results indicate that JOAB-40 is more potent than E121 in reducing colitis severity in mice and may be a promising future therapeutic target for the management of inflammatory bowel disease (IBD).

Pro-inflammatory Effects of Influenza Type A Virus PB1-F2 Protein-derived Peptide in Lipopolysaccharide-treated Macrophages.

Influenza A virus (IAV) has the potential to cause pandemics with considerable health and socio-economic burdens. A viral protein, polymerase basic 1- frame2 (PB1-F2), as a virulence factor, has pro-apoptotic activity and contributes to viral pathogenesis by delaying viral clearance and inducing inflammation. Macrophages are susceptible to IAV infection and produce high levels of inflammatory cytokines and chemokines. In the present study, the pro-inflammatory effects of PB1-F2 derived peptide was evaluated by measuring the expression of key inflammatory mediators in murine macrophage cell line J774.1. PB1-F2 treated macrophages were examined for nitric oxide (NO) production, inflammatory cytokines, and enzymes expression and pro-inflammatory cytokines secretion using Griess reagent, real-time polymerase chain reaction (PCR) and ELISA, respectively. Our results have shown that PB1-F2 peptide at non-cytotoxic concentrations (0.1-0.8 µmol/mL) had no effect on NO production. When applied to Lipopolysaccharide (LPS)-treated macrophages, PB1-F2 peptide at 0.8 μmol/mLincreasedinducible NO synthase (iNOS), cyclooxygenase (COX)-2, and interleukin (IL)-6 genes expression to 2.02, 3.81, and 3.65 folds, respectively. PB1-F2 at concentrations of 0.4 and 0.8 µm/mL increased tumor necrosis factor (TNF)-α transcription by 4.15 and 5.55 fold. At posttranslational level, TNF-α increased from 166.5±13.88 in LPS-treated cells to 773.6±95.27 and 1485±76.31 at concentrations of 0.4 and 0.8 μmol/mL in PB1-F2 peptide, respectively. However, PB1-F2 Peptide did not have any significant effect on IL-6 production. These findings suggest that PB1-F2 peptide may partly exert its enhancing role in viral pathogenicity through the induction of inflammatory mediators in macrophages. Hence, targeting PB1-F2 peptide would be helpful in the reduction of viral infection complications.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Alkylated/aminated nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates: Synthesis and anti-mycobacterial evaluation

The success in exploring anti-tubercular potency of nitroimidazole and quinoline, the core moieties of recently approved anti-tubercular drugs instigated us to synthesize a series of alkylated/aminated 2-methyl-5-nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates and to evaluate them for their activities against Mycobacterium tuberculosis as well as for their cytotoxicity towards the J774 murine macrophage cell line. Although the synthesized compounds did not surpass the activity of the standard drug Isoniazid, they have appreciable activities with minimal cytotoxicity. The synthesized nitroimidazole-7-chloroquinoline conjugate, 11c, having butyl chain as linker, proved to be the most potent among the series with an MIC50 value of 2.2 μg/mL.

Advanced glycation end products and lipopolysaccharides stimulate interleukin-6 secretion via the RAGE/TLR4-NF-κB-ROS pathways and resveratrol attenuates these inflammatory responses in mouse macrophages.

Macrophages are essential for regulating the physiology of pregnancy; however, excessive inflammatory responses to macrophages, induced by infection and/or endogenous danger signals, may potentially result in complications during pregnancy. Advanced glycation end-products (AGE) and lipopolysaccharides (LPS) are known to induce inflammation and are associated with adverse developmental outcomes. The aim of the present study was to examine the effect of AGE and LPS on cytokines in the J774 murine macrophage cell line and the potential effect of resveratrol on AGE- and LPS-induced inflammation in macrophages. AGE and LPS significantly increased IL-6 mRNA expression and secretion in J774 macrophages (P<0.05). Although AGE and LPS significantly stimulated IL-1β mRNA expression (P<0.05), they had no significant effect on IL-1β secretion. To assess the receptors for AGE and LPS, including receptor for AGE (RAGE) and Toll-like receptor (TLR4), blocking reagents (RAGE antagonist or TLR4 inhibitor) were added to the J774 macrophages. IL-6 secretion induced by AGE or LPS was significantly inhibited by pretreatment with RAGE antagonist (P<0.05) or TLR4 inhibitor (P<0.05). IL-6 secretion was dependent on nuclear factor (NF)-κB activation and the production of reactive oxygen species (ROS; P<0.05). Resveratrol suppressed mRNA expression and intracellular IL-6 production, resulting in significantly decreased IL-6 secretion after treatment with LPS or AGE (P<0.01). Furthermore, treatment with Ex527, which is a sirtuin-1 (SIRT1) inhibitor, significantly attenuated the anti-inflammatory effect of resveratrol (P<0.05), and treatment with 5-aminoimidazole-4-carboxamide ribonucleotide, which is a 5' adenosine monophosphate-activated protein kinase (AMPK) activator, resulted in a significant decrease in IL-6 secretion in J774 macrophages (P<0.05). The results of the present study indicated that AGE and LPS increase IL-6 secretion depending on NF-κB activation and ROS production through RAGE and/or TLR4 in the J774 murine macrophage cell line. Based on the present study, resveratrol appears to be an effective regulator of the inflammatory responses associated with SIRT1 and AMPK activation in macrophages. These results suggest that resveratrol may have therapeutic applications for controlling immune responses during pregnancy.

Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis.

Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 μg of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37°C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult. These findings provide promising evidence that assessment of individual phagocytic capacity can be made easier using this novel device.

Abstract 73: Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma

Abstract The transferrin receptor 1 (TfR1), also known as CD71, is a membrane glycoprotein involved in cellular iron uptake and regulation of cell growth. The high level of TfR1 expression on malignant cells and its key role in cancer cell pathology make this receptor an attractive target for antibody cancer therapy. We previously developed a mouse/human chimeric IgG3 specific for human TfR1 (ch128.1). This antibody exhibits direct cytotoxicity against certain human malignant B cells in vitro through TfR1 degradation and iron deprivation. Importantly, ch128.1 shows remarkable anti-tumor activity in xenograft models of disseminated multiple myeloma (MM) in immunosuppressed mice (SCID-Beige). Interestingly, this anti-tumor protection was observed even against MM cells (KMS-11 cells) that show no sensitivity to this antibody in vitro, suggesting the in vivo contributions of antibody effector functions. This possibility was supported by the lack of anti-tumor protection observed using a ch128.1 Fc mutant with impaired binding to FcγRs and to the complement component C1q. To examine host effector functions involved in ch128.1-mediated protection in our mouse model bearing KMS-11 tumors, depletion studies of complement and macrophages were performed. Complement depletion using cobra venom factor (CVF) did not affect protection, suggesting that complement-mediated cytotoxicity (CDC) is not a relevant mechanism of action. Notably, we now report that macrophage depletion using clodronate liposomes (clodrolip) significantly reduced protection, suggesting that these effector cells play a relevant role in the anti-tumor activity. Consistent with this result, we also report that ch128.1 is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) against KMS-11 cells in the presence of the murine macrophage cell line J774.2 or murine bone marrow-derived macrophages. To examine the role of iron deprivation in ch128.1-mediated tumor death in vivo, animals treated with ch128.1 were systemically supplemented with iron in a preliminary study. However, no difference in survival was observed, suggesting that iron deprivation is not a contributor to the effects of ch128.1 in our in vivo model or the iron dose tested was not optimal. Our results suggest that macrophages play a key role in ch128.1-mediated anti-tumor protection in our model and that ch128.1 can be an effective therapy of incurable human B-cell malignancies such as MM. Citation Format: Lai Sum Leoh, Yoon Kyung Kim, Pierre V. Candelaria, Otoniel Martínez-Maza, Tracy R. Daniels-Wells, Manuel L. Penichet. Role of macrophages in the antitumor activity of an anti-transferrin receptor 1 antibody ch128.1 in a xenograft model of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 73. doi:10.1158/1538-7445.AM2017-73

Surface engineering of Solid Lipid Nanoparticle assemblies by methyl α-d-mannopyranoside for the active targeting to macrophages in anti-tuberculosis inhalation therapy

This study describes the development of new mannosylated Solid Lipid Nanoparticle assemblies (SLNas) delivering rifampicin for an inhaled treatment of tuberculosis. SLNas were surface engineered with mannose residues to recognize mannose receptors located on infected alveolar macrophages and facilitate cell internalization. Two sets of SLNas were produced by the melt emulsifying technique using biocompatible lipid components, i.e. cholesteryl myristate combined with palmitic acid (PA set) or tripalmitin (TP set), in the presence of the targeting moiety, methyl α-d-mannopyranoside. Mannosylated SLNas were examined for their physical properties, drug payloads and release, as well as respirability in terms of emitted dose and respirable fraction determined by Next Generation Impactor. The most appropriate formulations were assessed for mannosylation using FTIR, XPS, SEM coupled with EDX analysis, and wettability assay, in comparison with the respective non-functionalized SLNas. Besides, cytotoxicity and cell internalization ability were established on J774 murine macrophage cell line. Mannosylated SLNas exhibited physical properties suitable for alveolar macrophage passive targeting, adequate rifampicin payloads (10–15%), and feasible drug maintenance within SLNas along the respiratory tract before macrophage internalization. Despite respirability impaired by powder cohesiveness, surface mannosylation provided quicker macrophage phagocytosis, giving evidence of an active targeting promotion.

In vivo/In vitro immune responses to L. major isolates from patients with no clinical response to Glucantime

Background:Leishmaniasis is a major health problem in some endemic areas of tropical and subtropical areas of the world. Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are essential cytokines associated with initiation of Th1 response. The main objective of this study was to evaluate of the type of immune response to L. major isolates from patients with no clinical response to antimonite (Glucantime).Materials and Methods:This experimental study was carried out during 2013–2014. In the current study Leishmania major were isolated from 10 CL patients with a history of at least one course of treatment with Meglumine antimonate (Sb5). The isolates were used to evaluate in vitro and in vivo response to Sb5. J774 murine macrophage cell line was used for in vitro tests and Balb/c mice was used for in vivo studies. IL-12 gene expression was evaluated using Real-time PCR and IFN-γ serum level was quantified using ELISA technique. SPSS (version: 20), analysis of Covariance (ANCOVA) was used for statistical analysis.Results:PCR results confirmed that all 10 isolates were L. major. The mean of IL-12 gene expression in vitro, in vivo and IFN-γ serum levels (pg/ml) after 2 and 3 weeks treatment in vivo, increased significantly following the treatment with Glucantime in the two groups of Balb/c mice infected either with patients' isolates or standard L. major. No significant difference was seen between the patients' isolates and standard species.Conclusions:Although the L. major were isolated from patients with active lesion and no clinical response to Glucantime after at least one courses of Glucantime treatment but in vivo and in vitro immune response of L. major isolates showed no difference between the patients' isolates and standard L. major.

Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells.

Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc2155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc2155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc2155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

Effect of SXWS/WSXWS peptides on chemotaxis and adhesion of the macrophage-like cell line J774.

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position "X" plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL-6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C-terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N-terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X.

Heterologous Expression of 3-O-Deacylase in Acinetobacter baumannii Modulates the Endotoxicity of Lipopolysaccharide

The lipopolysaccharide (LPS) of Acinetobacter baumannii is a potent stimulator of proinflammatory cytokines, such as interleukin-6 (IL-6). The 3-O-deacylase (PagL)-modifying enzyme that removes the 3-O-linked acyl chain from the disaccharide backbone of lipid A provides the opportunity to develop a new therapeutic compound that could reduce detrimental inflammatory responses. The plasmid pMMB66EH-PagL obtained by recombinant DNA technology was electroporated into A. baumannii ATCC 19606. Compared with wild-type LPS, outer membrane vesicles and inactivated whole cells of engineered bacteria had a statistically significant decreased ability to produce IL-6. Structural analysis of lipid A by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the profile of lipid A fractions under PagL expression was changed. Taken together, our data showed that recombinant penta-acylated lipid A had less immunoreactivity and that the tetra-acylated version of lipid A with TLR4 antagonist activity decreased the induction of IL-6 production in the murine macrophage cell line J774 A.1.

Deficient Beta-Mannosylation of Candida albicans Phospholipomannan Affects the Proinflammatory Response in Macrophages

Candida albicans produces a complex glycosphingolipid called phospholipomannan (PLM), which is present on the cell-wall surface of yeast and shed upon contact with host cells. The glycan moiety of PLM is composed of β-mannosides with degrees of polymerization up to 19 in C. albicans serotype A. PLM from serotype B strains displays a twofold decrease in the length of the glycan chains. In this study we compared the proinflammatory activities of PLMs purified from C. albicans serotype A and serotype B strains and from a bmt6Δ mutant of C. albicans, whose PLM is composed of short truncated oligomannosidic chain. We found that PLMs activate caspase-1 in murine macrophage cell line J774 independent of the glycan chain length although IL-1β secretion is more intense with long glycan chain. None of the tested PLMs stimulate ROS production, indicating that caspase-1 activation may occur through a ROS-independent pathway. On the other hand, only long-chain oligomannosides present on PLM from serotype A strain (PLM-A) are able to induce TNF-α production in macrophages, a property that is not affect by blocking endocytosis through latrunculin A treatment. Finally, we demonstrate that soluble and not cell surface-bound galectin-3, is able to potentiate PLM-A-induced TNF-α production in macrophages. PLMs from C. albicans serotype B and from bmt6∆ mutant are not able to induce TNF-α production and galectin-3 pretreatment does not interfere with this result. In conclusion, we show here that PLMs are able to evoke a proinflammatory state in macrophage, which is in part dependent on their glycosylation status. Long-glycan chains favor interaction with soluble galectin-3 and help amplify inflammatory response.

The Increases in mRNA Expressions of Inflammatory Cytokines by Adding Cleaning Solvent or Tetrachloroethylene in the Murine Macrophage Cell Line J774.1 Evaluated by Real-time PCR

The use of a petroleum-derived cleaning solvent for dry cleaning, instead of tetrachloroethylene (perchloroethylene, PCE), has increased. The cleaning solvent may induce immunological alteration. In this study, murine macrophage-lineage J774.1 cells were exposed to the cleaning solvent at 0, 25, 50, and 75 µg/ml or PCE at 0, 400, 600, 800, and 1,000 µg/ml by vigorous vortexing. Cell viability was determined. The mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-10, IL-12p40 (a dimer of IL-12), and IL-27p28 (a dimer of IL-27) were evaluated by real-time PCR. The mean viabilities in the 50 and 75 µg/ml groups of the cleaning solvent were significantly lower than that of the control. The mean mRNA expressions of TNF-α and IL-1β in the 50 µg/ml group were significantly higher than those in the control. For PCE, the mean viabilities at 600 µg/ml and over were significantly lower than that of the control. The mean expressions of IL-6 and IL-10 in the 800 µg/ml group were significantly higher than that in the control. The productions of IL-1β and TNF-α may be altered in human during intoxication of the cleaning solvent as well as those of IL-6 and IL-10 in human during that of PCE, and these may affect on immune cells.

Sevoflurane Combined with ATP Activates Caspase-1 and Triggers Caspase-1-Dependent Pyroptosis in Murine J774 Macrophages

Sevoflurane is one of the most commonly used volatile anesthetics. Recent studies have shown that sevoflurane plays an important role in modulation of inflammation and immunity. However, little is known about the related molecular mechanisms. This study was designed to investigate the effects and mechanisms of sevoflurane on inflammatory cell death pyroptosis in the murine macrophage cell line J774 cells. Sevoflurane combined with ATP could increase the level of activated caspase-1, pyroptosis, and reactive oxygen species (ROS). Furthermore, treatment of cells with the caspase-1 inhibitor Ac-YVAD-CMK dramatically decreased the percentage of pyroptosis. In addition, inhibition of ROS with N-acetyl-L-cysteine or diphenyleneiodonium significantly reduced the activated levels of caspase-1. These results demonstrated that sevoflurane combined with ATP could activate caspase-1 and trigger caspase-1-dependent pyroptosis through the modulation of ROS production.

Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology

BackgroundTheoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing.FindingsThe cycle threshold (Ct) values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure) varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p < 0.05).ConclusionIn this study, mycobacterial DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies.