Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.].

Staining of Lipid Droplets with Monodansylpentane.

Monodansylpentane (MDH) is a fluorophore that displays selective labeling of lipid droplets (LDs). The dye preferentially segregates into the neutral lipid cores of LDs and emits blue fluorescence, compatible with the simultaneous use of green and red fluorescent reporters in multi-color live-cell imaging. MDH can be used for visualizing LDs not only in cell cultures, but also in fixed tissues such as the fat body and ovaries from Drosophila. MDH is therefore a versatile marker for LDs in fluorescence microscopy.

Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

Monodansylpentane as a blue-fluorescent lipid-droplet marker for multi-color live-cell imaging.

Lipid droplets (LDs) are dynamic cellular organelles responsible for the storage of neutral lipids, and are associated with a multitude of metabolic syndromes. Here we report monodansylpentane (MDH) as a high contrast blue-fluorescent marker for LDs. The unique spectral properties make MDH easily combinable with other green and red fluorescent reporters for multicolor fluorescence imaging. MDH staining does not apparently affect LD trafficking, and the dye is extraordinarily photo-stable. Taken together MDH represents a reliable tool to use for the investigation of dynamic LD regulation within living cells using fluorescence microscopy.

A combination of the metabolic enzyme inhibitor APO866 and the immune adjuvant L-1-methyl tryptophan induces additive antitumor activity.

Many types of malignant cells have a higher nicotinamide adenine dinucleotide (NAD) turnover rate than normal cells, as well as the ability to escape immune responses. Indoleamine 2,3-dioxygenase (IDO) is reported to be a negative immune regulator. Overexpression of IDO in dendritic cells is observed in tumor-draining lymph nodes. IDO-expressing dendritic cells suppress T-cell activation and promote immune tolerance. The nicotinamide phosphoribosyl transferase (NAMPT) inhibitor APO866 (also called FK866 or WK175) selectively inhibits tumor growth through intracellular NAD depletion. The IDO-specific inhibitor L-1-methyl-tryptophan (L-1MT) activates immune responses and reduces tumor volume in murine tumor models. We combined L-1MT and APO866 treatments and tested their antitumor effects in the murine gastric and bladder tumor models. In immune-competent mice, a combination of APO866 and L-1MT had a better therapeutic effect than did either L-1MT or APO866 alone. The intracellular level of NAD was suppressed by APO866 but not L-1MT. However, an additive inhibitory effect on tumor growth was not observed in tumor-bearing immune-deficient mice. The new strategy of combining a metabolic inhibitor and an immune adjuvant induced a potent therapeutic effect.

A novel cancer therapy by skin delivery of indoleamine 2,3-dioxygenase siRNA.

PURPOSE: Indoleamine 2,3-dioxygenase (IDO), an enzyme that degrades tryptophan, is a negative immune regulatory molecule of dendritic cells. IDO-expressing dendritic cells suppress T cell responses and may be immunosuppressive in vivo. We hypothesized that silencing the IDO expression in skin dendritic cells in vivo could elicit antitumor activity in tumor-draining lymph nodes. EXPERIMENTAL DESIGN: The efficiency of IDO-specific small interfering RNA (siRNA) was evaluated in vitro and in vivo. The therapeutic effect was evaluated in MBT-2 murine bladder tumor model and CT-26 colon tumor models. RESULTS: IDO expression was down-regulated in CD11c-positive lymphocytes after IDO siRNA treatment. In vivo skin administration of IDO siRNA inhibited tumor growth and prolonged survival in both tumor models. The number of infiltrated T cells and neutrophils increased at tumor sites, which are correlated with therapeutic efficacy. The T cells may be mainly responsible for the immunologic rejection because the effect was abolished by depletion of CD8-positive T cells. Adoptive transfer of CD11c-positive dendritic cells from vaccinated mice delayed tumor progression. The cancer therapeutic effect was reproducibly observed with another IDO siRNA targeting at different site, suggesting the effect was not due to off-target effect. In a neu-overexpressing MBT-2 tumor model, IDO siRNA enhanced the therapeutic efficacy of Her2/Neu DNA vaccine. Down-regulation of IDO2, an IDO homologue, with siRNA also generated antitumor immunity in vivo. CONCLUSIONS: Antitumor immunity can be effectively elicited by physical delivery of siRNAs targeting immunoregulatory genes in skin dendritic cells in vivo, as shown by IDO and IDO2 in this report.

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model.

BACKGROUND: Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration. METHODS: In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine. RESULTS: The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicity T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunity as indicated by depletion of lymphocytes in vivo. CONCLUSION: Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.

Delivery of noncarrier naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer.

BACKGROUND: DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. The T helper type (Th)1 immune response is usually required for generating effective anti-tumor responses. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Using nozzle aerodynamics, a low pressure gene gun has been developed to decrease the noise associated with high pressure gene guns. Particles are propelled by supersonic flow through this novel nozzle. To test whether this gun could inoculate a DNA vaccine that stimulates an anti-tumor Th1 immune response, we examined the effect of direct delivery of naked DNA (i.e. without any carrier) on the anti-tumor immune response of mice. METHODS: The luciferase reporter plasmid DNA was delivered using a low-pressure biolistic device and expressed in C3H/HeN, BALB/c, and C57BL/6 mice. RESULTS: Plasmid DNA expression was mainly in the epidermis. Noncarrier naked neu DNA vaccine and gold particle-coated neu DNA vaccine (at 1 microg per mouse) had similar anti-tumor effects in C3H mice. However, cytokine profile examination showed the Th1-bias of the response induced by naked DNA vaccine and the Th2-bias of the response induced by coated DNA vaccine. CONCLUSIONS: A shift in the immune response to favour enhanced tumor rejection can be achieved by skin delivery of naked DNA vaccine.

The opposing effects of lipopolysaccharide on the antitumor therapeutic efficacy of DNA vaccine.

DNA vaccine represents a novel method to elicit immunity against infectious disease. Lipopolysaccharide (LPS) copurified with plasmid DNA may affect therapeutic efficacy and immunological response. We aimed to study the effect of LPS on the therapeutic efficacy of HER-2/neu DNA vaccine in a mouse tumor animal model. Plasmid DNA purified from commercial EndoFree plasmid purification kits functioned as a better therapeutic DNA vaccine than that purified from Non-EndoFree purification kit, which contains >or=0.5 microg LPS per 100 mg DNA plasmid. To further investigate the effect of LPS on the therapeutic efficacy of DNA vaccine, increasing amount of LPS was added to endotoxin-free plasmid DNA, and inoculated on mice with established tumors. One mug of LPS significantly attenuated the therapeutic effect of neu DNA vaccine and increased Th2 immune responses bias with interleukin-4 cytokine production. In contrast, high amount (100 microg) of LPS enhanced the therapeutic efficacy of neu DNA vaccine with an increase of cytotoxic T lymphocyte response and Th1 immune response. The effect of LPS on DNA vaccine was diminished when the tumor was grown in toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice. Our results indicate that variation in the LPS doses exerts opposing effects on the therapeutic efficacy of DNA vaccine, and the observed effect is TLR4 dependent.