J774 Mouse Macrophages Research Articles

Abstract 3048: The Role Of 17:0(2r-oh) Ceramide In Lps Activated Macrophages

Macrophages play a crucial role in the initiation and progression of atherosclerosis, contributing to the buildup of fatty deposits which results in the plaque formation in the walls of arteries. Macrophages modulate the mediators of inflammation and immune response in atherosclerosis. Bacterial lipopolysaccharide (LPS) exerts significant effects on these macrophages including secretion of pro-inflammatory cytokines. The current study is designed to examine the role of sphingosine intermediate N-(2'-(R)-hydroxyheptadecanoyl)-D-erythro-sphingosine (17:0(2R-OH) in the macrophage’s inflammation potential. Couple of studies have indicated a protective role for sphingosine however 17:0(2R-OH) didn’t receive adequate attention. Study design: Mouse J774 macrophages were cultured in Dulbecco’s Modified Eagle’s medium with 4.5g/L glucose and sodium pyruvate without L-glutamine containing 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C. The cells were treated in 6-well plates at a density of 5.5х10 6 cells/well. Sphingosine was dissolved in DMSO prior to the treatment. The cells were treated with the 17:0(2R-OH) with 3 different concentrations (5, 25, 50 μg) in duplicates with or without LPS except for the controls. The cells and media were collected at 3 time points (12,24,36) hours. Cells were harvested in Trizol reagent, RNA was then extracted from cells and genes expression were determined in RTPCR using SYBR Green. Results and Conclusion: Our initial microscopic observations suggest that 17:0(2R-OH) potentially modulates inflammation in macrophages, this appears to be dose-dependent. 17:0(2R-OH) has been identified in marine and freshwater fish, marine oils, seaweed, sea urchins, sea cucumbers and may have dietary benefits.

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.

Extracellular Vesicles Produced by the Cardiac Microenvironment Carry Functional Enzymes to Produce Lipid Mediators In Situ.

The impact of the polyunsaturated fatty acids (PUFAs) at physiological concentrations on the composition of eicosanoids transported within the extracellular vesicles (EVs) of rat bone marrow mesenchymal stem cells and cardiomyoblasts was reported by our group in 2020. The aim of this article was to extend this observation to cells from the cardiac microenvironment involved in the processes of inflammation, namely mouse J774 macrophages and rat heart mesenchymal stem cells cMSCs. Moreover, to enhance our capacity to understand the paracrine exchange between these orchestrators of cardiac inflammation, we investigated some machinery involved in the eicosanoid's synthesis transported by the EVs produced by these cells (including the two formerly described cells: bone marrow mesenchymal stem cells BM-MSC and cardiomyoblasts H9c2). We analyzed the oxylipin and the enzymatic content of the EVs collected from cell cultures supplemented (or not) with PUFAs. We prove that large eicosanoid profiles are exported in the EVs by the cardiac microenvironment cells, but also that these EVs carry some critical and functional biosynthetic enzymes, allowing them to synthesize inflammation bioactive compounds by sensing their environment. Moreover, we demonstrate that these are functional. This observation reinforces the hypothesis that EVs are key factors in paracrine signaling, even in the absence of the parent cell. We also reveal a macrophage-specific behavior, as we observed a radical change in the lipid mediator profile when small EVs derived from J774 cells were exposed to PUFAs. To summarize, we prove that the EVs, due to the carried functional enzymes, can alone produce bioactive compounds, in the absence of the parent cell, by sensing their environment. This makes them potential circulating monitoring entities.

Phenolic Compounds Known to Be Present in Lingonberry (Vaccinium vitis-idaea L.) Enhance Macrophage Polarization towards the Anti-Inflammatory M2 Phenotype.

Macrophages are pleiotropic immune cells whose phenotype can polarize towards the pro-inflammatory M1 or anti-inflammatory M2 direction as a response to environmental changes. In obesity, the number of macrophages in adipose tissue is enhanced, and they shift towards the M1 phenotype. Activated M1 macrophages secrete pro-inflammatory cytokines and adipokines involved in the development of systemic low-grade inflammation, complicating obesity. Polyphenols are widely found in the vegetable kingdom and have anti-inflammatory properties. We and others have recently found that lingonberry (Vaccinium vitis-idaea L.) supplementation is able to prevent the development of low-grade inflammation and its metabolic consequences in experimentally induced obesity. In the present study, we investigated the effects of twelve phenolic compounds known to be present in lingonberry (resveratrol, piceid, quercetin, kaempferol, proanthocyanidins, delphinidin, cyanidin, benzoic acid, cinnamic acid, coumaric acid, caffeic acid, and ferulic acid) on macrophage polarization, which is a meaningful mechanism determining the low-grade inflammation in obesity. Mouse J774 and human U937 macrophages and commercially available phenolic compounds were used in the studies. Three of the twelve compounds investigated showed an effect on macrophage polarization. Resveratrol, kaempferol, and proanthocyanidins enhanced anti-inflammatory M2-type activation, evidenced as increased expression of Arg-1 and MRC-1 in murine macrophages and CCL-17 and MRC-1 in human macrophages. Resveratrol and kaempferol also inhibited pro-inflammatory M1-type activation, shown as decreased expression of IL-6, NO, and MCP-1 in murine macrophages and TNF-α and IL-6 in human macrophages. In the further mechanistic studies, the effects of the three active compounds were investigated on two transcription factors important in M2 activation, namely on PPARγ and STAT6. Resveratrol and kaempferol were found to enhance PPARγ expression, while proanthocyanidins increased the phosphorylation of STAT6. The results suggest proanthocyanidins, resveratrol, and kaempferol as active constituents that may be responsible for the positive anti-inflammatory effects of lingonberry supplementation in obesity models. These data also extend the previous knowledge on the anti-inflammatory effects of lingonberry and encourage further studies to support the use of lingonberry and lingonberry-based products as a part of a healthy diet.

FTY720 Reduces Lipid Accumulation by Upregulating ABCA1 through Liver X Receptor and Sphingosine Kinase 2 Signaling in Macrophages

Formation of foam cells as a result of excess lipid accumulation by macrophages is a pathological hallmark of atherosclerosis. Fingolimod (FTY720) is an immunosuppressive agent used in clinical settings for the treatment of multiple sclerosis and has been reported to inhibit atherosclerotic plaque development. However, little is known about the effect of FTY720 on lipid accumulation leading to foam cell formation. In this study, we investigated the effects of FTY720 on lipid accumulation in murine macrophages. FTY720 treatment reduced lipid droplet formation and increased the expression of ATP-binding cassette transporter A1 (ABCA1) in J774 mouse macrophages. FTY720 also enhanced the expression of liver X receptor (LXR) target genes such as FASN, APOE, and ABCG1. In addition, FTY720-induced upregulation of ABCA1 was abolished by knockdown of sphingosine kinase 2 (SphK2) expression. Furthermore, we found that FTY720 treatment induced histone H3 lysine 9 (H3K9) acetylation, which was lost in SphK2-knockdown cells. Taken together, FTY720 induces ABCA1 expression through SphK2-mediated acetylation of H3K9 and suppresses lipid accumulation in macrophages, which provides novel insights into the mechanisms of action of FTY720 on atherosclerosis.

Eicosapentaenoic acid (EPA, c20:5 n-3) increased ABCA1-mediated cholesterol efflux from J774 mouse macrophages or THP-1 human macrophages by modulating secreted eicosanoids

Background and Aims : A diet rich in eicosapentaenoic acid (EPA, C20:5 n-3) is cardioprotective. We studied in vitro the impact of EPA membrane incorporation on the anti-atherogenic ABCA1 efflux pathway in the absence or presence of LPS to create the inflammatory microenvironment observed within atherosclerotic plaques.

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.

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Atherogenic oxoaldehyde of cholesterol induces innate immune response in monocytes and macrophages.

Cholesterol oxidation product, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (cholesterol 5,6-secosterol, ChSeco or Atheronal-A), formed at inflammatory sites, has been shown to promote monocyte differentiation into macrophages and cause elevated expression of macrophage scavenger receptors. Since inflammation is a key event at all stages of atherosclerotic plaque formation, the pro-inflammatory actions of ChSeco in human THP-1 monocytes and mouse J774 macrophages were investigated in the present study by employing ELISA, qRT-PCR, and functional assays. An increase in the secretion of interleukin-8 and platelet-derived growth factor (PDGF) isoform AA and, to a limited extent, PDGF isoform BB was observed into the culture medium of THP-1 monocytes exposed to ChSeco. However, no changes were seen in the secretion of tumor necrosis factor-alpha. In J774 macrophages treated with ChSeco, there was an upregulation of gene expression of several pro-inflammatory cytokines and their receptors. Concomitantly, there was down-regulation of gene expression of interleukin-1ß, interleukin-10, and lymphotoxin-beta. An increase in the release of interleukin-18 and chemokine (C-C motif) ligand-20 from J774 macrophages (which corroborated well with their gene expression profiles) and increased binding of THP-1 monocytes to ChSeco-exposed human aortic endothelial cells were observed. The results of the present study, for the first time, demonstrate the pro-inflammatory action of ChSeco and suggest the underlying pro-atherogenic mechanisms. These could be mediated through enhanced monocyte recruitment into the sub-endothelial space and subsequent proliferation of smooth muscle cells under the influence of monocyte-derived PDGF.

Suppressors of Cytokine Signaling (SOCS)1 and SOCS3 Proteins Are Mediators of Interleukin-10 Modulation of Inflammatory Responses Induced by Chlamydia muridarum and Its Major Outer Membrane Protein (MOMP) in Mouse J774 Macrophages.

The immunopathology of chlamydial diseases is exacerbated by a broad-spectrum of inflammatory mediators, which we reported are inhibited by IL-10 in macrophages. However, the chlamydial protein moiety that induces the inflammatory mediators and the mechanisms by which IL-10 inhibits them are unknown. We hypothesized that Chlamydia major outer membrane protein (MOMP) mediates its disease pathogenesis, and the suppressor of cytokine signaling (SOCS)1 and SOCS3 proteins are mediators of the IL-10 inhibitory actions. Our hypothesis was tested by exposing mouse J774 macrophages to chlamydial stimulants (live Chlamydia muridarum and MOMP) with and without IL-10. MOMP significantly induced several inflammatory mediators (IL-6, IL-12p40, CCL5, CXCL10), which were dose-dependently inhibited by IL-10. Chlamydial stimulants induced the mRNA gene transcripts and protein expression of SOCS1 and SOCS3, with more SOCS3 expression. Notably, IL-10 reciprocally regulated their expression by reducing SOCS1 and increasing SOCS3. Specific inhibitions of MAPK pathways revealed that p38, JNK, and MEK1/2 are required for inducing inflammatory mediators as well as SOCS1 and SOCS3. Chlamydial stimulants triggered an M1 pro-inflammatory phenotype evidently by an enhanced nos2 (M1 marker) expression, which was skewed by IL-10 towards a more M2 anti-inflammatory phenotype by the increased expression of mrc1 and arg1 (M2 markers) and the reduced SOCS1/SOCS3 ratios. Neutralization of endogenously produced IL-10 augmented the secretion of inflammatory mediators, reduced SOCS3 expression, and skewed the chlamydial M1 to an M2 phenotype. Inhibition of proteasome degradation increased TNF but decreased IL-10, CCL5, and CXCL10 secretion by suppressing SOCS1 and SOCS3 expressions and dysregulating their STAT1 and STAT3 transcription factors. Our data show that SOCS1 and SOCS3 are regulators of IL-10 inhibitory actions, and underscore SOCS proteins as therapeutic targets for IL-10 control of inflammation for Chlamydia and other bacterial inflammatory diseases.

Zinc Increases ABCA1 by Attenuating Its Clearance Through the Modulation of Calmodulin Activity.

Aim: We previously revealed that Ca++-activated calmodulin binds to ABCA1 by the region near the PEST sequence and retards its calpain-mediated degradation to increase HDL biogenesis. Calmodulin activity is reportedly modulated also by other nutritional divalent cations; thus, we attempted to determine whether Zn++ is involved in the regulation of ABCA1 stability through the modulation of calmodulin activity.Methods: The effects of Zn++ on ABCA1 expression was investigated in J774 mouse macrophage cell-line cells and HepG2 human hepatoma cell-line cells.Results: Zn++ increased ABCA1 expression, not by increasing the mRNA but by attenuating its decay rate, more prominently in the presence of cAMP. Accordingly, it enhanced cell cholesterol release with extracellular apolipo-protein A-I. Calmodulin binding to ABCA1 was increased by Zn++ and Ca++. Zn++ suppressed calpain-mediated hydrolysis of the peptide of ABCA1 cytosolic loop, including the PEST sequence and the calmodulin-binding site, in a calmodulin-dependent fashion, in the presence of the minimum amount of Ca++ to activate calpain, but not calmodulin. Calpain activity was not directly inhibited by Zn++ at the concentration for enhancing calmodulin binding to ABCA1.Conclusion: Nutritional divalent cation Zn++ is involved in the regulation of ABCA1 activity and biogenesis of HDL through the modulation of calmodulin activity. The results were consistent with previous clinical findings that Zn++ increased plasma HDL in the conditions of sympathetic activation, such as type 2 diabetes and chronic hemodialysis.

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.

Cellular pharmacokinetics and intracellular activity of the bacterial fatty acid synthesis inhibitor, afabicin desphosphono against different resistance phenotypes of Staphylococcus aureus in models of cultured phagocytic cells

Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log10 after incubation in broth for 30h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log10) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S.aureus and remains fully active against strains resistant to these comparators.

Contrasting effects of membrane enrichment with polyunsaturated fatty acids on phospholipid composition and cholesterol efflux from cholesterol-loaded J774 mouse or primary human macrophages

A high consumption of polyunsaturated fatty acids (PUFAs), particularly n-3 PUFAs, is atheroprotective. PUFAs incorporation into membrane phospholipids alters the functionality of membrane proteins. We studied the consequences of the in vitro supplementation of several PUFAs on the FA profiles and on ABCA1-dependent cholesterol efflux capacities from cholesterol-loaded macrophages. Arachidonic acid (AA, C20:4 n-6) and, to a lesser extent, eicosapentaenoic acid (EPA, C20:5 n-3), dose-dependently impaired cholesterol efflux from cholesterol-loaded J774 mouse macrophages without alterations in ABCA1 expression, whereas docosahexaenoic acid (DHA, C22:6 n-3) had no impact. AA cells exhibited higher proportions of arachidonic acid and adrenic acid (C22:4 n-6), its elongation product. EPA cells exhibited slightly higher proportions of EPA associated with much higher proportions of docosapentaenoic acid (C22:5 n-3), its elongation product and with lower proportions of AA. Conversely, both EPA and DHA and, to a lesser extent, AA decreased cholesterol efflux from cholesterol-loaded primary human macrophages (HMDM). The differences observed in FA profiles after PUFA supplementations were different from those observed for the J774 cells. In conclusion, we are the first to report that AA and EPA, but not DHA, have deleterious effects on the cardioprotective ABCA1 cholesterol efflux pathway from J774 foam cells. Moreover, the membrane incorporation of PUFAs does not have the same impact on cholesterol efflux from murine (J774) or human (HMDM) cholesterol-loaded macrophages. This finding emphasizes the key role of the cellular model in cholesterol efflux studies and may partly explain the heterogeneous literature data on the impact of PUFAs on cholesterol efflux.

Abstract 1459: Construction and assessment of a novel Listeria monocytogenes-based vaccine targeting squamous cell carcinoma

Abstract Introduction: Immunotherapy represents a potential therapeutic option for many tumors including squamous cell carcinoma (SCC). L. monocytogenes is a potent delivery vehicle that can elicit cellular immunity specific for the encoded antigens, and overcome the immunosuppressive cancer environment. Here we test the hypothesis that L. monocytogenes strain lacking 2 secreted chaperones (ΔprsA2 ΔhtrA) would be able to induce a specific immune response against SCC and would represent a safe vaccine due to its hyper-attenuation. Methods: Recombinant L. monocytogenes ΔprsA2 ΔhtrA strains were engineered to express Cyp2e1 squamous carcinoma antigen using standard molecular techniques. All genes were verified by DNA sequencing, and secreted proteins were assessed by western blot. Bacterial growth assays were tested in broth and intracellularly in J774 mouse macrophage cells. The safety of the vaccine strains were tested in vivo by counting the CFUs in target organs and measuring the liver function tests in serum. The efficacy was then assessed in a subcutaneous mouse model of tumor growth together with L. monocytogenes ΔactA prfA* strain and ΔprsA2 ΔhtrA strain with no tumor antigen. Mice were first challenged with KLN205 tumor cells, and then vaccinated with 3 immunizations weekly. The tumors volumes were recorded with a caliper every 3 days and organs weights were measured at the end of the experiment. Results: The Cyp2e1 antigen was detectable in L. monocytogenes supernatant fractions, and the secretion of tumor antigens did not impact the growth of mutant strains in broth culture. Bacterial replication within infected macrophage cells showed defective intracellular growth for L. monocytogenes ΔprsA ΔhtrA expressing tumor antigen while L. monocytogenes ΔactA prfA strains exhibited modest levels of inhibition in comparison to wild type. Mice infected with ΔprsA2 ΔhtrA strains had no CFUs recovered from liver or spleen while half of the mice infected with ΔactA prfA strains exhibited bacterial burdens within the liver and no bacteria were recovered from the spleen. Consistently, all the infected mice exhibited normal liver function as measured by AST and ALT levels. The results confirm the safety of ΔprsA2 ΔhtrA strains in vivo. Additionally, ΔprsA2 ΔhtrA vaccine strains significantly halted the SCC growth when compared to the control mice. Preliminary data suggests that tumor antigen-specific differences in tumor size and spleens weights could be visualized between the groups of mice immunized with L.monocytogenes recombinant strains expressing Cyp2e1 antigen versus no antigen. These data suggest that the highly attenuated L. monocytogenes ΔprsA2 ΔhtrA strains may indeed be efficient in stimulating SCC regression. Conclusion: L. monocytogenes ΔprsA2 ΔhtrA strains represent a novel and safe vaccine that altered the immunosuppressive microenvironment of SCC and inhibited tumor growth. Citation Format: Alyaa F. Hessin, Nancy E. Freitag. Construction and assessment of a novel Listeria monocytogenes-based vaccine targeting squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1459.

ApoAI-derived peptide increases glucose tolerance and prevents formation of atherosclerosis in mice

Aims/hypothesisFinding new treatment alternatives for individuals with diabetes with severe insulin resistance is highly desired. To identify novel mechanisms that improve glucose uptake in skeletal muscle, independently from insulin levels and signalling, we have explored the therapeutic potential of a short peptide sequence, RG54, derived from apolipoprotein A-I (ApoA-I).MethodsINS-1E rat clonal beta cells, C2C12 rat muscle myotubes and J774 mouse macrophages were used to study the impact of RG54 peptide on glucose-stimulated insulin secretion, glucose uptake and cholesterol efflux, respectively. GTTs were carried out on diet-induced insulin-resistant and Leprdb diabetic mouse models treated with RG54 peptide, and the impact of RG54 peptide on atherosclerosis was evaluated in Apoe−/− mice. Control mice received ApoA-I protein, liraglutide or NaCl.ResultsThe synthetic RG54 peptide induced glucose uptake in cultured muscle myotubes by a similar amount as insulin, and also primed pancreatic beta cells for improved glucose-stimulated insulin secretion. The findings were verified in diet-induced insulin-resistant and Leprdb diabetic mice, jointly confirming the physiological effect. The RG54 peptide also efficiently catalysed cholesterol efflux from macrophages and prevented the formation of atherosclerotic plaques in Apoe−/− mice.Conclusions/interpretationThe RG54 peptide exhibits good prospects for providing glucose control and reducing the risk of cardiovascular disease in individuals with severe insulin resistance.

Pharmacokinetics/pharmacodynamics of lefamulin in a neutropenic murine pneumonia model with Staphylococcus aureus and Streptococcus pneumoniae.

ObjectivesTo present results of preclinical studies that supported further development of lefamulin for treating patients with community-acquired bacterial pneumonia (CABP).MethodsThe effect of bovine lung surfactant on the antibacterial activity of lefamulin against Streptococcus pneumoniae and Staphylococcus aureus was determined by broth microdilution assay. In vitro accumulation of lefamulin was evaluated in J774 mouse macrophages. Pharmacokinetics was assessed in female BALB/c (Bagg albino) mice treated with subcutaneous lefamulin (35 or 70 mg/kg). In neutropenic lung infection experiments, BALB/c mice received intraperitoneal cyclophosphamide before challenge with single S. pneumoniae or S. aureus strains; subcutaneous lefamulin (1.25–160 mg/kg) was given twice daily post-infection. Hill models described relationships between AUC/MIC ratios and changes in log10 cfu.ResultsLung surfactant did not significantly increase lefamulin MIC values against test strains. Lefamulin uptake in macrophages was rapid (a plateau was reached in ∼3 h). In mice, distribution of lefamulin [plasma to epithelial lining fluid (ELF)] was rapid, showing an ∼2-fold increase in lefamulin exposure in the ELF during the 5.5 h period. Median plasma AUC/MIC ratios associated with 1 and 2 log10 cfu reductions from baseline were 1.37 and 2.15, respectively, for S. pneumoniae and 2.13 and 6.24 for S. aureus. Corresponding ELF results were 14.0 and 22.0 for S. pneumoniae and 21.7 and 63.9 for S. aureus.ConclusionsOverall, lefamulin displays desirable pharmacokinetic/pharmacodynamic relationships that are predictive of the clinical effectiveness of lefamulin and other antibacterial agents used to treat CABP.

Glucose-6-phosphate dehydrogenase (G6PD) activity can modulate macrophage response to Leishmania major infection

Glucose-6-phosphate dehydrogenase (G6PDH) ultimately plays a critical role in macrophage functions used against infectious agents. The present study investigated whether changes in G6PDH activity could influence the resistance of infected macrophages against Leishmania major infection. Mouse peritoneal and J774 macrophages were infected, respectively, ex vivo and in vitro, with L. major and then exposed to an inhibitor (6-aminonicotinamide) or activator (LPS + melatonin) of G6PDH activity for 24 h. Cell viability [using MTT assay] was measured to assess any direct toxicity from the doses of inhibitor/activator used for the macrophage treatments. Nitric oxide (NO) produced by the cells and released into culture supernatants was measured (Griess method) and cell G6PDH activity was also determined. Moreover, the number of amastigotes form Leishmania in macrophages that developed over a 7-d period was evaluated. The results showed that an increase in G6PDH activity after treatment of both types of macrophages with a combination of LPS + melatonin caused significant increases in NO production and cell resistance against L. major amastigote formation/survival. However, exposure to 6-aminonicotinamide led to remarkable suppression of G6PDH activity and NO production, events that were associated with a deterioration in cell resistance against (and an increase in cell levels of) the parasites. The results suggested that activation or suppression of G6PDH activity could affect leishmanicidal function of both mouse peritoneal and J774 macrophages. Thus, regulation of macrophages via modulation of G6PDH activity appears to provide a novel window for those seeking to develop alternative therapies for the treatment of leishmaniasis.

Investigation of Legionella contamination in soil

Objective To understand the contamination of Legionella in soil and the molecular type, sensitivity to antibiotics and intracellular growth ability of isolated strains. Methods A total of 123 soil samples from public places in Beijing were collected. Legionella in soil was detected by GVPC (Legionella selective medium) culture and quantitative real-time PCR detection. The intracellular growth ability of the Legionella isolates were tested by cell culture methods with mouse macrophage J774. The genotypes of the isolates were determined by pulsed-field gel electrophoresis. Antibiotic sensitivity detection of the isolates was conducted with E-test. Results Quantitative PCR assay detected Legionella in flower & bird market samples and reservoir samples in summer. The positive rates were 50.00% and 24.24% respectively. The plate culture method only detected Legionella in reservoir samples in summer. The positive rate was 6.06%. All the two Legionella strains isolated, TR1 and TR2, were L. pneumophila, and the serotypes were type 5 and type 8 respectively. All the two strains of L. pneumophila had high intracellular growth ability. PFGE typing showed two different band types. The drug sensitivity test showed that the two strains were sensitive to erythromycin, azithromycin, rifampicin, levofloxacin and moxifloxacin. TR1 showed resistance to cefuroxime, and TR2 was sensitive to cefuroxime. Conclusions Legionella pneumophila was detected in the soil of public places, and the isolates have higher intracellular growth ability, indicating the pathogenicity of the strains and suggesting the potential of causing legionellosis epidemic. Therefore, we should strengthen the monitoring of Legionella in public places.