DNA Methylation Maintains Allele-specific KIR Gene Expression in Human Natural Killer Cells

Killer immunoglobulin-like receptors (KIR) bind self–major histocompatibility complex class I molecules, allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes, but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression, 3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5′ gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor, 5-aza-2′-deoxycytidine, induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus, NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles

Circumventing AKT-Associated Radioresistance in Oral Cancer by Novel Nanoparticle-Encapsulated Capivasertib

Background: Development of radioresistance in oral squamous cell carcinoma (OSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in recent decades. Effective strategies to overcome radioresistance are necessary to improve the therapeutic outcomes of radiotherapy in OSCC patients. Methods: Cells and xenograft tumors were irradiated using the Small Animal Radiation Research Platform. AKT inhibitor capivasertib (AZD5363) was encapsulated into cathepsin B-responsible nanoparticles (NPs) for tumor-specific delivery. Cell viability was measured by alamarBlue, cell growth was determined by colony formation and 3D culture, and apoptosis was assessed by flow cytometry with the staining of Fluorescein isothiocyanate (FITC) Annexin V and PI. An orthotopic tongue tumor model was used to evaluate the in vivo therapeutic effects. The molecular changes induced by the treatments were assessed by Western blotting and immunohistochemistry. Results: We show that upregulation of AKT signaling is the critical mechanism for radioresistance in OSCC cells, and AKT inactivation by a selective and potent AKT inhibitor capivasertib results in radiosensitivity. Moreover, relative to irradiation (IR) alone, IR combined with the delivery of capivasertib in association with tumor-seeking NPs greatly enhanced tumor cell repression in 3D cell cultures and OSCC tumor shrinkage in an orthotopic mouse model. Conclusions: These data indicate that capivasertib is a potent agent that sensitizes radioresistant OSCC cells to IR and is a promising strategy to overcome failure of radiotherapy in OSCC patients

Pdgfrα-Cre mediated knockout of the aryl hydrocarbon receptor protects mice from high-fat diet induced obesity and hepatic steatosis.

Aryl hydrocarbon receptor (AHR) agonists such as dioxin have been associated with obesity and the development of diabetes. Whole-body Ahr knockout mice on high-fat diet (HFD) have been shown to resist obesity and hepatic steatosis. Tissue-specific knockout of Ahr in mature adipocytes via adiponectin-Cre exacerbates obesity while knockout in liver increases steatosis without having significant effects on obesity. Our previous studies demonstrated that treatment of subcutaneous preadipocytes with exogenous or endogenous AHR agonists disrupts maturation into functional adipocytes in vitro. Here, we used platelet-derived growth factor receptor alpha (Pdgfrα)-Cre mice, a Cre model previously established to knock out genes in preadipocyte lineages and other cell types, but not liver cells, to further define AHR's role in obesity. We demonstrate that Pdgfrα-Cre Ahr-floxed (Ahrfl/fl) knockout mice are protected from HFD-induced obesity compared to non-knockout Ahrfl/fl mice (control mice). The Pdgfrα-Cre Ahrfl/fl knockout mice were also protected from increased adiposity, enlargement of adipocyte size, and liver steatosis while on the HFD compared to control mice. On a regular control diet, knockout and non-knockout mice showed no differences in weight gain, indicating the protective phenotype arises only when animals are challenged by a HFD. At the cellular level, cultured cells from brown adipose tissue (BAT) of Pdgfrα-Cre Ahrfl/fl mice were more responsive than cells from controls to transcriptional activation of the thermogenic uncoupling protein 1 (Ucp1) gene by norepinephrine, suggesting an ability to burn more energy under certain conditions. Collectively, our results show that knockout of Ahr mediated by Pdgfrα-Cre is protective against diet-induced obesity and suggest a mechanism by which enhanced UCP1 activity within BAT might confer these effects

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

<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

LAMP1 decreased within MoDCs infected with <i>P. gingivalis</i> expressing Mfa1.

<p><b>A)</b> Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs. <b>B)</b> The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* <i>p</i><0.001). <b>C)</b> The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson’s correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* <i>p</i><0.0001) (Table <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004647#ppat.1004647.t004" target="_blank">4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004647#ppat.1004647.t005" target="_blank">5</a>). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons.</p

Low LC3-II expression in MoDCs infected with <i>P. gingivalis</i> expressing Mfa1.

<p><b>A)</b> Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. <b>B)</b> The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons. <b>C)</b> Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. <b>D)</b> LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons.</p

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

DC-SIGN mRNA expression in MoDCs at 12 hours.

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