Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing's sarcoma cells

Purpose: The present in vitro study was conducted to evaluate the effects of the histone deacetylase inhibitors (HDIs) suberoyl anilide hydroxamic acid (SAHA), sodium butyrate (NaB) and MS-275 applied as single agents or in combination with TRAIL in Ewing's sarcoma. Methods: Cytotoxic activities were assessed by cytofluorometric analysis of propidium iodide uptake, DNA fragmentation and mitochondrial depolarisation as well as by measuring caspase-9 and -3 activities. Cell-surface expression of TRAIL receptors was determined by cytofluorometry, and histone H4 acetylation was assessed by western blot. Results: All three HDIs potently induced cell death in the two cell lines explored, SK-ES-1 and WE-68. However, they seemed to differ in their modes of action. SAHA and NaB induced mitochondrial depolarisation as well as caspase-9 and -3 activities, and their cytotoxic effects could be significantly reduced by the pan-caspase inhibitor z-VAD-fmk. MS-275 was a much weaker inducer of caspase-9 and -3 activities as well as mitochondrial injury; consistently, z-VAD-fmk had little effect on MS-275-mediated activities. Combined treatment of HDIs and TRAIL led to an additive effect in SK-ES-1 cells and a supra-additive effect in WE-68 cells. Yet, HDIs did not increase cell-surface expression of TRAIL receptor 2, but rather decreased it. Selective inhibition of caspase-8 in WE-68 cells and HDI treatment of CADO-ES-1 cells, a Ewing's sarcoma cell line deficient in caspase-8 expression, revealed that caspase-8 was not required for HDI-mediated apoptosis. Conclusions: These results suggest that HDIs may be considered as a novel treatment strategy for Ewing's sarcoma either applied as monotherapy or in combination with TRAIL

Deletion of Arginase 2 Ameliorates Retinal Neurodegeneration in a Mouse Model of Multiple Sclerosis

Optic neuritis is a major clinical feature of multiple sclerosis (MS) and can lead to temporary or permanent vision loss. Previous studies from our laboratory have demonstrated the critical involvement of arginase 2 (A2) in retinal neurodegeneration in models of ischemic retinopathy. The current study was undertaken to investigate the role of A2 in MS-mediated retinal neuronal damage and degeneration. Experimental autoimmune encephalomyelitis (EAE) was induced in wild-type (WT) and A2 knockout (A2(-/-)) mice. EAE-induced motor deficits, loss of retinal ganglion cells, retinal thinning, inflammatory signaling, and glial activation were studied in EAE-treated WT and A2(-/-) mice and their respective controls. Increased expression of A2 was observed in WT retinas in response to EAE induction. EAE-induced motor deficits were markedly reduced in A2(-/-) mice compared with WT controls. Retinal flat mount studies demonstrated a significant reduction in the number of RGCs in WT EAE retinas in comparison with normal control mice. A significant improvement in neuronal survival was evident in retinas of EAE-induced A2(-/-) mice compared with WT. RNA levels of the proinflammatory molecules CCL2, COX2, IL-1alpha, and IL-12alpha were significantly reduced in the A2(-/-) EAE retinas compared with WT EAE. EAE-induced activation of glia (microglia and Muller cells) was markedly reduced in A2(-/-) retinas compared with WT. Western blot analyses showed increased levels of phospho-ERK1/2 and reduced levels of phospho-BAD in the WT EAE retina, while these changes were prevented in A2(-/-) mice. In conclusion, our studies establish EAE as an excellent model to study MS-mediated retinal neuronal damage and suggest the potential value of targeting A2 as a therapy to prevent MS-mediated retinal neuronal injury

<i>Porphyromonas gingivalis</i> Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

<div><p>Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of <i>Porphyromonas gingivalis</i> in human monocyte-derived DCs (MoDCs). We employed a panel of <i>P. gingivalis</i> isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+<i>P. gingivalis</i> strains by MoDCs resulted in lower intracellular killing and higher intracellular content of <i>P. gingivalis</i>. Moreover, Mfa1+<i>P. gingivalis</i> was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+<i>P. gingivalis</i> strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, <i>P. gingivalis</i> uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+<i>P. gingivalis</i> promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.</p></div

TLRs activation restores LC3-II expression and inhibits the growth of Mfa1⁺Pg within human MoDCs.

<p><b>A)</b> Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (<i>E. coli</i> LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour. <b>B)</b> Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&2 ligands (<i>E. coli</i> LPS and Pam3csk4) <b>C)</b> The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** <i>p</i><0.001).</p

Mfa1⁺Pg up-regulate the expression of DC-SIGN in human MoDCs.

<p><b>A)</b> DC-SIGN mRNA expression in <i>P. gingivalis</i>-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2^-(ΔΔCt) method. The statistical analysis was performed using the <i>t-test</i>, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* <i>p</i><0.001). <b>B)</b> Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1⁺Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1⁺Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. <b>C)</b> Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1⁺Pg and FimA⁺Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* <i>p</i><0.01).</p

DC-SIGN mRNA expression in MoDCs at 12 hours.

<p>DC-SIGN mRNA expression in MoDCs at 12 hours.</p

Induction of autophagy impairs the survival of Mfa1⁺Pg strain within MoDCs.

<p><b>A)</b> Survival of <i>P. gingivalis</i> strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show <i>P. gingivalis</i> strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1⁺Pg, Pg381 and FimA⁺Pg survival within MoDCs are shown in figures <b>B, C and D</b> respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1⁺Pg, Pg381 and FimA⁺Pg) between treated (rapamycin) and untreated were significantly different overtime (<i>p-value</i> <0.=001).</p

Pearson’s co-localization of Rab5 (red) and P. gingivalis (green) signals.

<p>Pearson’s co-localization of Rab5 (red) and P. gingivalis (green) signals.</p

Low LC3-II signals in human MoDCs infected with Mfa1⁺Pg.

<p><b>A)</b> Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1⁺Pg, FimA⁺Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of <i>P. gingivalis</i> and LC3-II showed in the right panels. <b>B)</b> Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons of three different experiments (* <i>p</i><0.001, # <i>p</i><0.01). <b>C)</b> Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* <i>p</i><0.001, # <i>p</i><0.01).</p

Lower intracellular content of Mfa1⁺Pg in MoDCs treated with Rapamycin.

<p><b>A)</b> Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* <i>P</i><0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey’s test for multiple comparisons. <b>B)</b> Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after <i>P. gingivalis</i> infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections). <b>C)</b> Quantifications of the fluorescent intensity of CFSE-labeled <i>P. gingivalis</i> and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons (* <i>P</i><0.001).</p