Murine Macrophage-like Cell Line Research Articles

Elucidation of regulatory mechanisms in the activation of dendritic cells by combined stimuli with PRR ligands and sulfated polysaccharide fucoidan

Abstract Fucoidan, a series of natural high-molecular weight sulfated polysaccharides derived from brown algae, has been reported to have various physiological effects such as antitumor, antiviral, and immunomodulatory activities. In a previous study, we demonstrated that fucoidan from Cladosiphon okamuranus (Okinawamozuku) was effectively activate murine macrophage-like cell line RAW264 in synergistic action with dectin-1-stimulating beta-glucan from Saccharomyces cerevisiae. In this study, we further examined their synergistic mode of action of Okinawamozuku-derived fucoidan in cooperate with pathogen stimulation in terms of dendritic cell activation. After myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were differentiated by culturing bone marrow cells isolated from C57BL/6J mice with Flt3 ligand, cytokine production upon stimulation with fucoidan and various pathogen components (pattern recognition receptor, PRR ligands) was measured. As the results, fucoidan alone enhanced the production of interferon (IFN)-gamma by the Flt3-L-induced mixed BMDCs, and besides, fucoidan synergistically augmented IFN-gamma production with Pam3CSK4 (bacterial TLR1/TLR2 ligand) or Poly(I:C)/LyoVec (viral RIG-I ligand). On the other hand, their IFN-alpha productions were reinforced with Poly(I:C) (viral TLR3 ligand), ODN1585 (viral TLR9 ligand) and Poly(I:C)/LyoVec, although no synergic enhancement by fucoidan was observed. These results suggested that fucoidan was extremely effective in activating dendritic cells, which play a central role in the regulation of immune function to eliminate infectious pathogens. Supported by grants from JSPS KAKENHI (JP21K05423)

Use of the quantum dot-labeled solid lipid nanoparticles for delivery of streptomycin and hydroxychloroquine: A new therapeutic approach for treatment of intracellular Brucella abortus infection

Brucellosis is considered one of the most important infectious diseases affecting any tissue and organ in the human body. Due to the intracellular pathogenesis of Brucella species, the use of conventional antibiotics for managing chronic brucellosis has several limitations. Therefore, the study focused on the use of solid lipid nanoparticles (SLN) to deliver streptomycin (STR) for intracellular infection, with or without the combination of hydroxychloroquine (HCQ) to evaluate if there might be a boost in the antibiotic effect when using the STR or STR-NPs alone. We used the double emulsion technique to synthesize Nano drug carriers; afterward, the physicochemical characteristics of synthesized Nano drug carriers were determined. The in vitro antibacterial activity of free drugs and Nano drug carriers were evaluated using well diffusion, broth microdilution assays (BMD), and murine macrophage-like cells cell line J774A.1. Additionally, acute and chronic phases of brucellosis were inducted into Wistar rats, and healing capacity of Nano drug carriers on liver and spleen tissues was compared with free drugs. The zeta potential of nanoparticles, means of size, Polydispersity Index (PDI), drugs loading, and encapsulation efficiency were 15.2 mV, 312.5 ± 26 nm, 0.433 ± 0.09, 16.6% and 89.5%, respectively. Well diffusion and BMD methods did not show a significantly differ between free drugs and nano drug carriers. However, the Nano drug carriers remarkably decreased the number of bacteria in the cell line compared to the free drugs. STR/HCQ-SLN enhanced the healing processes of the liver and spleen after brucellosis induction. STR/HCQ-SLN showed better inhibitory effects against the chronic phase of B. abortus infection in comparison to the STR-SLN, but this difference was not statistically significant. Using nanoplatforms to enhance conventional anti-brucellosis agents is promising, green and safe. Due to the continuous release of drugs, drugs increase their accumulation at the site of infection, causing a more significant effect on the chronic and acute phases of brucellosis.

Pal Affects the Proliferation in Macrophages and Virulence of Brucella, and as Mucosal Adjuvants, Provides an Effective Protection to Mice Against Salmonella Enteritidis.

The purpose of this study was to elucidate the roles of peptidoglycan-associated lipoprotein (Pal protein) in the proliferation of Brucella in macrophage and bacterial virulence, and to evaluate the immune effect of Pal protein to Salmonella enteritidis. Murine macrophage-like cell line Raw264.7 was stimulated by recombinant Pal protein, and the expression of TNF-α and IFN-γ were up-regulated, but not it of IL-1β and IL-6. The macrophages infection and in vitro simulated stress assays showed that deletion of pal gene reduced the proliferation of Brucella in macrophages, the survival in acidic, oxidative and polymyxin B-contained environment. The mice infection assay showed that mice challenged with the pal mutant strain were found to have more severe splenomegaly, but less bacterial load. After oral immunization of mice, Pal protein induced a higher titer of mucosal and humoral antibody (IgA and IgG) against heat-killed Salmonella enteritidis, and a stronger Th1 cellular immune response. The challengte experiments showed Pal protein elevated the survival rate and reduced the bacterial load of spleens in immunized mice. In conclusion, our results revealed the important roles of pal gene in Brucella virulence, and Pal protein was a potentially valuable adjuvant against mucosal pathogens, such as Salmonella enteritidis.

Protection against Lipopolysaccharide-Induced Endotoxemia by Terrein Is Mediated by Blocking Interleukin-1β and Interleukin-6 Production.

Terrein is a fungal metabolite and has been known to exert anti-melanogenesis, anti-cancer, and anti-bacterial activities. However, its role in endotoxemia has never been investigated until now. In the present study, we examined the effect of terrein on lipopolysaccharide (LPS)-induced endotoxemia in mice and characterized the potential mechanisms of action. Treatment with terrein increased the survival of mice and decreased the production of inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6) in an LPS-induced endotoxemia model. In addition, terrein suppressed the LPS-induced production of IL-1β and IL-6 in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expression of IL-1β and IL-6 was also inhibited by terrein in LPS-stimulated RAW 264.7 cells. Further study demonstrated that terrein blocked LPS-induced phosphorylation of p65 subunit of nuclear factor (NF)/κB and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) was also suppressed by terrein treatment. Collectively, these results suggest that terrein exerts a protective effect again LPS-induced endotoxemia in mice by blocking the production of inflammatory cytokines. Our results also suggest that the anti-inflammatory effect of terrein might be mediated, at least in part, by blocking the activation of NF-κB, JNK, and p38 MAPK signaling pathways.

The anti-inflammatic activity of a sulfated polysaccharide Fucoidan in innate immune cells

Abstract Fucoidan is a series of natural sulfated polysaccharides derived from brown seaweeds and have reported to possess various biological activities such as anti-tumor, anti-virus, and immuno-modulatory effects. In previous study, we revealed that fucoidan derived from Cladosiphon okamuranus activated murine macrophage-like cell line RAW264 cooperatively with Zymosan, a Saccharomyces cerevisiae-derived β-glucan. On the other hand, several reports demonstrated that fucoidan inhibited lipopolysaccharide (LPS)-induced production of nitrogen oxide (NO) and inflammatory cytokines by RAW264 cells. Therefore, in this study, we investigated the potential of fucoidan derived from Undaria pinnatifida to regulate the excessive activation of RAW264 cells in response to overstimulation with pathogen components. RAW264 cells were inoculated at 20000 cells/well in 96-well culture plates, and after a recovery culture for 24 hr, the cells were treated with fucoidan with or without appropriate concentrations of each ligand for pattern recognition receptors (PRRs) including Toll-like receptors (TLRs). Whereas the production of NO was significantly enhanced with fucoidan alone, it was dose-dependently inhibited by fucoidan under stimulation with Pam3CSK4 (TLR1/TLR2 ligand), heat killed Listeria monocytogenes (HKLM, TLR2 ligand), LPS (TLR4 ligand), Pam2CGDPKHPKSF (FSL-1, TLR2/TLR6 ligand). These results suggested that fucoidan have beneficial ability to alleviate excessive inflammatory reaction during pathogenic infection and maintain suitable immune balance. Supported by Laboratory of Analytical Food Immunoscience belonging YM is a donated fund laboratory established in Faculty of Agriculture, Kyushu University by Ventuno Co., Ltd.

Phytochemical Constituents of Aquilaria malaccensis Leaf Extract and Their Anti-Inflammatory Activity against LPS/IFN-γ-Stimulated RAW 264.7 Cell Line.

This study aims to identify the major phytochemical constituents in Aquilaria malaccensis (Thymelaeaceae) ethanolic leaf extract (ALEX-M) and elucidate their ability to suppress nitric oxide (NO) production from a murine macrophage-like cell line (RAW 264.7) stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Dichloromethane (DCM) and ethyl acetate (EtOAc) fractions of ALEX-M were subjected to column chromatography. Eight known compounds were isolated for the first time from this species. Compounds were identified using spectroscopic techniques (IR, UV, HRESIMS, and 1D and 2D NMR). Anti-inflammatory activity of both extract and isolated compounds were investigated in vitro. The fractions offered the isolation of epifriedelanol (1), 5-hydroxy-7,4′-dimethoxyflavone (2), luteolin-7,3′,4′-trimethyl ether (3), luteolin-7,4′-dimethyl ether (4), acacetin (5), aquilarinenside E (6), iriflophenone-2-O-α-l-rhamnopyranoside (7), and iriflophenone-3-C-β-glucoside (8). The findings suggest the pharmacological potential of the crude extract (ALEX-M) and its isolates as natural anti-inflammatory agents, capable of suppressing NO production in RAW 264.7 cells stimulated by LPS/IFN-γ.

Design and Characterization of a Recombinant Brucella abortus RB51 Vaccine That Elicits Enhanced T Cell-Mediated Immune Response.

Brucella abortus vaccines help control bovine brucellosis. The RB51 strain is a live attenuated vaccine with low side effects compared with other live attenuated brucellosis vaccines, but it provides insufficient protective efficacy. Cell-mediated immune responses are critical in resistance against intracellular bacterial infections. Therefore, we hypothesized that the listeriolysin O (LLO) expression of Listeria monocytogenes, BAX, and SMAC apoptotic proteins in strain RB51 could enhance vaccine efficacy and safety. B. abortus RB51 was transformed separately with two broad-host-range plasmids (pbbr1ori-LLO and pBlu–mLLO-BAX-SMAC) constructed from our recent work. pbbr1ori-LLO contains LLO, and pBlu–mLLO-BAX-SMAC contains the mutant LLO and BAX-SMAC fusion gene. The murine macrophage-like cell line J774A.1 was infected with the RB51 recombinant strain containing pBlu-mLLO-BAX-SMAC, RB51 recombinant strain containing LLO, and RB51 strain. The bacterial cytotoxicity and survival and apoptosis of host cells contaminated with our two strain types—RB51 recombinants or the parental RB51—were assessed. Strain RB51 expressing mLLO and BAX-SMAC was tested in BALB/c mice and a cell line for enhanced modulation of IFN-γ production. LDH analysis showed that the RB51-mLLO-BAX-SMAC and RB51-LLO strains expressed higher cytotoxicity in J774A.1 cells than RB51. In addition, RB51 recombinants had lower macrophage survival rates and caused higher levels of apoptosis and necrosis. Mice vaccinated with the RB51 recombinant containing mLLO-BAX-SMAC showed an enhanced Th1 immune response. This enhanced immune response is primarily due to bacterial endosome escape and bacterial antigens, leading to improved apoptosis and cross-priming. This potentially enhanced TCD8+- and T cell-mediated immunity leads to the increased safety and potency of the RB51 recombinant (RB51 mLLO-BAX-SMAC) as a vaccine candidate against B. abortus.

Lactobacillus paracasei KW3110 Prevents Inflammatory-Stress-Induced Mitochondrial Dysfunction in Mouse Macrophages

Lactobacillus paracasei KW3110 (KW3110) has anti-inflammatory effects, including the prevention of blue light exposure induced retinal inflammation and ageing-related chronic inflammation in mice. The mechanism involves the promotion of anti-inflammatory cytokine interleukin (IL)-10 production by KW3110, leading to reduced pro-inflammatory cytokine IL-1β production. Although various stress-induced mitochondrial damages are associated with excessive inflammatory responses, the effect of KW3110 on inflammatory-stress-induced mitochondrial damage remains unknown. In this study, we investigated the effect of KW3110 on inflammatory stress-induced mitochondrial damage using the murine macrophage-like cell line J774A.1. KW3110 treatment suppressed lipopolysaccharide (LPS)-induced mitochondrial dysfunction, including downregulation of membrane potential, induction of reactive oxygen species, and respiratory dysfunction. In addition, KW3110 prevented LPS-induced disruption of mitochondrial morphology including cristae structures. IL-10 treatment also ameliorated LPS-induced mitochondrial dysfunction and morphology disruption. These results suggest that KW3110 prevents LPS-induced mitochondrial dysfunction, potentially via promoting IL-10 production in mouse macrophages. We are the first to reveal a suppressive effect of lactic acid bacteria on mitochondrial morphology disruption in inflammatory-stressed macrophages. Our findings contribute to understanding inflammatory-stress-induced mitochondrial damage and developing food ingredients with preventive effects on mitochondrial-damage-derived inflammatory conditions.

Terpolymer-chitosan membranes as biomaterial.

The present study shows a novel copolymer synthesis, its application in the membrane design and the physicochemical and biological characterization of the biomaterial obtained. Terpolymer starting diisopropyl fumarate (F), vinyl benzoate (V) and 2-hydroxyethyl methacrylate (H) was prepared by thermal radical polymerization. This polymer (FVH) was obtained in several monomer ratios and characterized by spectroscopic and chromatographic methods (FTIR, 1 H-NMR and SEC). The best relationship of F:V:H was 5:4:1, which allows efficient interaction with chitosan through cross-linking with borax to achieve scaffolds for potential biomedical applications. The membranes were obtained by solvent casting and analyzed by scanning electron microscopy (SEM), swelling behavior and mechanical properties. In addition, we studied the possible cytotoxicity and biocompatibility of these materials using a murine macrophage-like cell line (RAW 264.7) and bone marrow mesenchymal progenitor cells (BMPC), respectively, taking into account their intended applications. The results of this study show that the terpolymer obtained and its combination with a natural polymer is a very interesting strategy to obtain a biomaterial with possible applications in regenerative medicine and this could be extended to other structurally related systems.

Activin a inhibits foam cell formation and up-regulates ABCA1 and ABCG1 expression through Alk4-Smad signaling pathway in RAW 264.7 macrophages

BackgroundActivin A has been reported to play important roles in the pathogenesis of atherosclerosis. The purpose of this study is to investigate the effects of activin A on oxidized low-density lipoprotein (ox-LDL)-induced foam cell formation and explore the underlying molecular mechanisms in murine macrophage-like cell line RAW 264.7. MethodsThe effects of activin A on Dil-labeled ox-LDL uptake were examined by confocal microscopy and flow cytometry analysis. The mRNA and protein levels of cholesterol receptors were analyzed by RT-qPCR and western blot analysis, respectively. To investigate whether activin receptor-like kinase 4 (Alk4) is required for activin A-mediated cellular effects, cells were pre-treated with SB-431542. The involvement of Smad2, Smad3 and Smad4 was confirmed by transfection with specific small interfering RNAs (siRNAs). ResultsActivin A inhibits ox-ldl-induced foam cell formation and class A scavenger receptors (SR‐A) expression, while up-regulates ATP-binding cassette transporter A1 (ABCA1) and ABCG1 expression in RAW 264.7 macrophages. Pre-treatment with SB-431542 abolished activin A-mediated anti-atherogenic effect. Knockdown of Smad2 reversed activin A-induced inhibition of ox-LDL uptake and SR-A expression. However, knockdown of Smad3 or Smad4 did not have such effect. Meanwhile, knockdown of either Smad2, Smad3 or Smad4 reversed the activin A-induced up-regulation of ABCA1 and ABCG1. ConclusionsOur study provides novel evidence that activin A may exert anti-atherogenic effects through Alk4-Smad signaling pathway in RAW 264.7 macrophages.

Genome-guided investigation of anti-inflammatory sesterterpenoids with 5-15 trans-fused ring system from phytopathogenic fungi.

Fungal terpenoids catalyzed by bifunctional terpene synthases (BFTSs) possess interesting bioactive and chemical properties. In this study, an integrated approach of genome mining, heterologous expression, and in vitro enzymatic activity assay was used, and these identified a unique BFTS sub-clade critical to the formation of a 5-15 trans-fused bicyclic sesterterpene preterpestacin I (1). The 5-15 bicyclic BFTS gene clusters were highly conserved but showed relatively wide phylogenetic distribution across several species of the diverged fungal classes Dothideomycetes and Sordariomycetes. Further genomic organization analysis of these homologous biosynthetic gene clusters from this clade revealed a glycosyltransferase from the graminaceous pathogen Bipolaris sorokiniana isolate BS11134, which was absent in other 5-15 bicyclic BFTS gene clusters. Targeted isolation guided by BFTS gene deletion led to the identification of two new sesterterpenoids (4, and 6) from BS11134. Compounds 2 and 4 showed moderate effects on LPS-induced nitrous oxide production in the murine macrophage-like cell line RAW264.7 with in vitro inhibition rates of 36.6 ± 2.4% and 24.9 ± 2.1% at 10 μM, respectively. The plausible biosynthetic pathway of these identified compounds was proposed as well. This work revealed that phytopathogenic fungi can serve as important sources of active terpenoids via systematic analysis of the genomic organization of BFTS biosynthetic gene clusters, their phylogenetic distribution in fungi, and cyclization properties of their metabolic products. KEY POINTS: • Genome mining of the first BFTS BGC harboring a glycosyltransferase. • Gene-deletion guided isolation revealed three novel 5-15 bicyclic sesterterpenoids. • Biosynthetic pathway of isolated sesterterpenoids was proposed.

Statins attenuate antiviral IFN‐β and ISG expression via inhibition of IRF3 and JAK/STAT signaling in poly(I:C)‐treated hyperlipidemic mice and macrophages

Viral infection is a significant burden to health care worldwide. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, are widely used as cholesterol-lowering drugs. Recently, long-term statin therapy was shown to reduce the antiviral immune response; however, the underlying molecular mechanisms are unclear. Here, we found that simvastatin decreased polyinosinic-polycytidylic acid [poly(I:C)]-induced expression of antiviral interferon (IFN)-β and IFN-stimulated genes (ISGs) in the bronchoalveolar lavage fluid (BALF) and lungs of mice with high-fat diet-induced hyperlipidemia. Macrophages were the dominant cell type in the BALF of poly(I:C)-treated mice. We examined the effects of simvastatin in primary lung macrophages and found that simvastatin suppressed poly(I:C)-induced expression of IFN-β and ISGs. We examined the molecular mechanisms of statin-mediated inhibition of antiviral gene expression using murine macrophage-like cell line, J774.1/JA-4. Simvastatin and pitavastatin decreased poly(I:C)-induced expression of IFN-β and ISGs. Moreover, they repressed poly(I:C)-induced phosphorylation of IFN regulatory factor (IRF) 3 and signal transducers and activators of transcription (STAT) 1, which is involved in Janus kinase (JAK)/STAT signaling. Mevalonate and geranylgeranyl pyrophosphate (GGPP), but not cholesterol, counteracted the negative effect of statins on IFN-β and ISG expression and phosphorylation of IRF3 and STAT1. The geranylgeranyltransferase inhibitor suppressed poly(I:C)-induced expression of IFN-β and ISGs and phosphorylation of IRF3 and STAT1. These results suggest that statins suppressed the expression of IFN-β and ISGs in poly(I:C)-treated hyperlipidemic mice and murine macrophages and that these effects occurred through the inhibition of IRF3 and JAK/STAT signaling in macrophages. Furthermore, GGPP recovered the statin-suppressed IRF3 and JAK/STAT signaling in poly(I:C)-treated macrophages.

In vitro evaluation of antileishmanial activity of copper (I) complexes

In the research for the development of new drugs for the therapy of American tegumentary leishmaniasis, copper has been studied for its antileishmania activity. This study aims to report the activity of three copper(I) complexes on parasites of the species L. amazonensis and L. guyanensis. The metal complexes were tested according to in vitro antileishmanial assays, against promastigote and amastigote forms of the most prevalent species in the state of Amazonas, Brazil. Cytotoxicity of the complexes was evaluated in murine macrophage-like cell line (MJ774). The results of the in vitro assays indicated that, among the copper complexes tested, the homoleptic phosphine complex [Cu(thp)4][PF6](thp=tris-hydroxymethylphosphine) presented promising activity against the evolutionary forms of L. amazonensis, and obtained a IC50 of 26.45 and 24.61 µM in a period of 48 and 72 h, respectively. The results for copper complex at concentration 160 µM in amastigote forms showed a decrease in the infection index (32% of infected cells) and, in the cytotoxicity assay with MJ774, 52.43% of cell viability was observed. The results showed that the complex [Cu(thp)4][PF6] presented significant biological activity, indicating a need for future in vivo studies.

Immunomodulation of J774A.1 Murine Macrophages by Lactiplantibacillus plantarum Strains Isolated From the Human Gastrointestinal Tract and Fermented Foods.

Lactobacillus plantarum species (recently re-named Lactiplantibacillus (Lpb.) plantarum subsp. plantarum) can be isolated from both either the mammalian gut or specific fermented foods where they may be present at high concentrations. Whilst Lpb. plantarum strains have been proposed as potential probiotic candidates, the ability of resident strains consumed in fermented foods to interact with the host is unclear. The main objective of this study was to investigate the cellular location and ability of three different food-borne Lpb. plantarum strains isolated from different sources (table olives and cheese) to modulate the immune response of a murine macrophage-like cell line (J774A.1). For that purpose, macrophages were exposed to the three different Lpb. plantarum strains for 24 h and the expression of a panel of genes involved in the immune response, including genes encoding pattern-recognition receptors (TLRs and NLRs) and cytokines was evaluated by qRT-PCR. We also utilized chemical inhibitors of intracellular pathways to gain some insight into potential signaling mechanisms. Results showed that the native food strains of Lpb. plantarum were able to modulate the response of J774A.1 murine macrophages through a predominately NOD signaling pathway that reflects the transient intracellular location of these strains within the macrophage. The data indicate the capacity of food-dwelling Lpb. plantarum strains to influence macrophage-mediated host responses if consumed in sufficient quantities.

Modification of human monocytes and macrophages by magnetic nanoparticles in vitro for cell-based delivery

The aim of the study was to develop a method for the modification of human monocytes/macrophages by iron oxide magnetic nanoparticles in vitro . Materials and methods . Iron oxide magnetic nanoparticles were obtained by a co-precipitation method and coated with a thin SiO 2 layer and polyethylene glycol 3000. Murine macrophage-like cell line RAW 264.7, primary human monocytes and macrophages were incubated with magnetic nanoparticles for 1–24 hours. The efficiency of cellular uptake of nanoparticles was measured using a ferrozine-based method and microcopy with Perls’ Prussian blue staining. The cell viability was tested by fluorescent flow cytometry using SYTOX Green. Results. Incubation of RAW264.7 cell, human monocytes and macrophages with magnetic nanoparticles at a concentration ˃ 5 µg/mL on a rotator for 1 hour at 37 °С provides the loading of nanoparticles into > 99% of cells. The magnetic nanoparticles have no adverse effect on the cell viability. The RAW264.7 cells modified with nanoparticles showed no change in migration activity. The efficiency of the nanoparticle uptake by macrophages was ˃50 pkg (Fe)/cell. Conclusion . According to the proposed method, macrophages loaded with magnetic nanoparticles have proved viable, they retain the ability to migrate, and therefore can be used as cell-based delivery systems for tumor diagnostic and therapy.

Evolution of Antibiotic Resistance in Surrogates of Francisella tularensis (LVS and Francisella novicida): Effects on Biofilm Formation and Fitness.

Francisella tularensis, the causative agent of tularemia, is capable of causing disease in a multitude of mammals and remains a formidable human pathogen due to a high morbidity, low infectious dose, lack of a FDA approved vaccine, and ease of aerosolization. For these reasons, there is concern over the use of F. tularensis as a biological weapon, and, therefore, it has been classified as a Tier 1 select agent. Fluoroquinolones and aminoglycosides often serve as the first line of defense for treatment of tularemia. However, high levels of resistance to these antibiotics has been observed in gram-negative bacteria in recent years, and naturally derived resistant Francisella strains have been described in the literature. The acquisition of antibiotic resistance, either natural or engineered, presents a challenge for the development of medical countermeasures. In this study, we generated a surrogate panel of antibiotic resistant F. novicida and Live Vaccine Strain (LVS) by selection in the presence of antibiotics and characterized their growth, biofilm capacity, and fitness. These experiments were carried out in an effort to (1) assess the fitness of resistant strains; and (2) identify new targets to investigate for the development of vaccines or therapeutics. All strains exhibited a high level of resistance to either ciprofloxacin or streptomycin, a fluoroquinolone and aminoglycoside, respectively. Whole genome sequencing of this panel revealed both on-pathway and off-pathway mutations, with more mutations arising in LVS. For F. novicida, we observed decreased biofilm formation for all ciprofloxacin resistant strains compared to wild-type, while streptomycin resistant isolates were unaffected in biofilm capacity. The fitness of representative antibiotic resistant strains was assessed in vitro in murine macrophage-like cell lines, and also in vivo in a murine model of pneumonic infection. These experiments revealed that mutations obtained by these methods led to nearly all ciprofloxacin resistant Francisella strains tested being completely attenuated while mild attenuation was observed in streptomycin resistant strains. This study is one of the few to examine the link between acquired antibiotic resistance and fitness in Francisella spp., as well as enable the discovery of new targets for medical countermeasure development.

DUSP5 (dual-specificity protein phosphatase 5) suppresses BCG-induced autophagy via ERK 1/2 signaling pathway

Autophagy is considered as an effective strategy for host cells to eliminate intracellular Mycobacterium tuberculosis (Mtb). Dual-specificity phosphatase 5 (DUSP5) is an endogenous phosphatase of ERK1/2, and plays an important role in host innate immune responses, its function in autophagy regulation however remains unexplored. In the present study, the function of DUSP5 in autophagy in Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-infected RAW264.7 cells, a murine macrophage-like cell line, was examined by assessing the alteration of the cell morphology, expression of autophagy markers, and ERK1/2 signaling activation. The results demonstrated that the BCG infection could induce DUSP5 expression and activate ERK1/2 signaling in RAW264.7 cells; an activation of ERK1/2 signaling contributed to autophagic process in RAW264.7 cells. Moreover, DUSP5 knockdown increased the expression of autophagy-related proteins (Atgs), including LC3-II, Beclin1, Atg5 and Atg7. However, an overexpression of DUSP5 exhibited an opposite effect. Mechanistically, DUSP5 could inhibit the formation of autophagosome by suppressing the phosphorylation of signaling molecules in ERK1/2 signaling cascade. This study thus demonstrated a novel role of DUSP5 in modulating autophagy inRAW264.7 cells in response to BCG infection in particular, and autophagy macrophage to Mtb in general.

Systematic evaluation of potential pathogenicity of Salmonella Indiana

Salmonella Indiana has emerged in recent years as an important zoonotic pathogen, but its pathogenicity has not been fully elucidated. In this study, using in vivo and in vitro animal and cellular experimental model systems, we evaluated the pathogenicity of Salmonella Indiana (S. Indiana) compared with three other serotypes of Salmonella, S. Enteritidis, S. Typhimurium and S. Thompson. The animal experiments included observations of clinical symptoms, pathological changes and determination of median lethal dose in mice. The adhesion and invasiveness and intracellular proliferative capacity of Salmonella in vitro were measured with the murine macrophage-like cell line RAW264.7 cells and the human colon adenocarcinoma cell line Caco-2 cells. The results of animal experiments showed that S. Indiana, S. Enteritidis, S. Typhimurium and S. Thompson caused histopathological changes in most organs to varying degrees, primarily in the liver and intestine of mice. The gross lesions included white necrotic foci on the liver surface with different levels. The histopathological changes of monocyte/macrophage infiltration and coagulative necrosis were observed in the liver. Intestinal villi became short and were sloughed off, and lymphocyte infiltration was found in the submucosa. Compared with the other serotypes, the pathological changes caused by S. Indiana were slighter and had a relatively high median lethal dose in mice. The results of adhesion and invasion tests showed that the intracellular growth trend of most Salmonella strains was positively correlated with the number of pathogens adhering to and invading cells. Compared with the strains of the other three serotypes, most S. Indiana strains exhibited significantly lower adhesion and invasiveness to RAW264.7 and Caco-2 cells within 30 min. Most S. Indiana strains displayed twice to four times lower intracellular proliferation within 24 h in RAW264.7 cells. In conclusion, S. Indiana was pathogenic, but its pathogenicity was lower than that of S. Typhimurium and S. Enteritidis, and was similar to that of S. Thompson.

Inhibition of Skin Inflammation by Scytonemin, an Ultraviolet Sunscreen Pigment.

Scytonemin is a yellow-green ultraviolet sunscreen pigment present in different genera of aquatic and terrestrial blue-green algae, including marine cyanobacteria. In the present study, the anti-inflammatory activities of scytonemin were evaluated in vitro and in vivo. Topical application of scytonemin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear swelling in BALB/c mice. The expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) was also suppressed by scytonemin treatment in the TPA-treated ear of BALB/c mice. In addition, scytonemin inhibited lipopolysaccharide (LPS)-induced production of TNF-α and nitric oxide (NO) in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expressions of TNF-α and iNOS were also suppressed by scytonemin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced NF-κB activity was significantly suppressed by scytonemin treatment in RAW 264.7 cells. Our results also showed that the degradation of IκBα and nuclear translocation of the p65 subunit were blocked by scytonemin in LPS-stimulated RAW 264.7 cells. Collectively, these results suggest that scytonemin inhibits skin inflammation by blocking the expression of inflammatory mediators, and the anti-inflammatory effect of scytonemin is mediated, at least in part, by down-regulation of NF-κB activity. Our results also suggest that scytonemin might be used as a multi-function skin care ingredient for UV protection and anti-inflammation.

Isorhamnetin, a 3'-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner.

Lysosome is the principal organelle for the ultimate degradation of cellular macromolecules, which are delivered through endocytosis, phagocytosis, and autophagy. The lysosomal functions have been found to be impaired by fatty foods and aging, and more importantly, the lysosomal dysfunction in macrophages has been reported as a risk of atherosclerosis development. In this study, we searched for dietary polyphenols which possess the activity for enhancing the lysosomal degradation in J774.1, a murine macrophage-like cell line. Screening test utilizing DQ-BSA digestion identified isorhamnetin (3'-O-methylquercetin) as an active compound. Interestingly, structural comparison to inactive flavonols revealed that the chemical structure of the B-ring moiety in isorhamnetin is the primary determinant of its lysosome-enhancing activity. Unexpectedly isorhamnetin failed to inhibit mTORC1-TFEB signaling, a master regulator of lysosomal biogenesis and function. Our data suggested that the other molecular mechanism might be critical for the regulation of lysosomes in macrophages.Abbreviations: ANOVA: analysis of variance; ApoE: apolipoprotein E; ATP6V0D2: ATPase H+ transporting V0 subunit d2; BAF: bafilomycin A1; BODIPY: boron dipyrromethene; BSA: bovine serum albumin; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified eagle medium; DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LC-MS/MS: liquid chromatography tandem mass spectrometry; MITF: microphthalmia-associated transcription factor; MRM: multiple reaction monitoring; mTORC1: mechanistic target of rapamycin complex 1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor γ; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SDS: sodium dodecyl sulfate; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TBS: Tris-buffered saline; TFA: trifluoroacetic acid; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcriptional factor EB; TFEC: transcription factor EC; V-ATPase: vacuolar-type proton ATPase.