Notch3-regulated microRNAs impair CXCR4-dependent maturation of thymocytes allowing maintenance and progression of T-ALL.

Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.

SCF and IL-33 regulate mouse mast cell phenotypic and functional plasticity supporting a pro-inflammatory microenvironment.

Mast cells (MCs) are multifaceted innate immune cells often present in the tumor microenvironment (TME). Several recent findings support their contribution to the transition from chronic inflammation to cancer. However, MC-derived mediators can either favor tumor progression, inducing the spread of the tumor, or exert anti-tumorigenic functions, limiting tumor growth. This apparent controversial role likely depends on the plastic nature of MCs that under different microenvironmental stimuli can rapidly change their phenotype and functions. Thus, the exact effect of unique MC subset(s) during tumor progression is far from being understood. Using a murine model of colitis-associated colorectal cancer, we initially characterized the MC population within the TME and in non-lesional colonic areas, by multicolor flow cytometry and confocal microscopy. Our results demonstrated that tumor-associated MCs harbor a main connective tissue phenotype and release high amounts of Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α. This MC phenotype correlates with the presence of high levels of Stem Cell Factor (SCF) and IL-33 inside the tumor. Thus, we investigated the effect of SCF and IL-33 on primary MC cultures and underscored their ability to shape MC phenotype eliciting the production of pro-inflammatory cytokines. Our findings support the conclusion that during colonic transformation a sustained stimulation by SCF and IL-33 promotes the accumulation of a prevalent connective tissue-like MC subset that through the secretion of IL-6 and TNF-α maintains a pro-inflammatory microenvironment.

Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

Energy Transfer from Magnetic Iron Oxide Nanoparticles: Implications for Magnetic Hyperthermia.

Magnetic iron oxide nanoparticles (IONPs) have gained momentum in the field of biomedical applications. They can be remotely heated via alternating magnetic fields, and such heat can be transferred from the IONPs to the local environment. However, the microscopic mechanism of heat transfer is still debated. By X-ray total scattering experiments and first-principles simulations, we show how such heat transfer can occur. After establishing structural and microstructural properties of the maghemite phase of the IONPs, we built a maghemite model functionalized with aminoalkoxysilane, a molecule used to anchor (bio)molecules to oxide surfaces. By a linear response theory approach, we reveal that a resonance mechanism is responsible for the heat transfer from the IONPs to the surroundings. Heat transfer occurs not only via covalent linkages with the IONP but also through the solvent hydrogen-bond network. This result may pave the way to exploit the directional control of the heat flow from the IONPs to the anchored molecules-i.e., antibiotics, therapeutics, and enzymes-for their activation or release in a broader range of medical and industrial applications.

Iron Oxide Nanoparticles with and without Cobalt Functionalization Provoke Changes in the Transcription Profile via Epigenetic Modulation of Enhancer Activity.

Despite the progress in the field of nanotoxicology, much about the cellular mechanisms that mediate the adverse effects of nanoparticles (NPs) and, in particular, the possible role of epigenetics in nanotoxicity, remains to be clarified. Therefore, we studied the changes occurring in the genome-wide distribution of H3K27ac, H3K4me1, H3K9me2, and H3K27me3 histone modifications and compared them with the transcriptome after exposing NIH3T3 cells to iron-based magnetic NPs (i.e., Fe2O3 and Fe2O3@Co NPs). We found that the transcription response is mainly due to changes in the genomic distribution of H3K27ac that can modulate the activity of enhancers. We propose that alteration of the epigenetic landscape is a key mechanism in defining the gene expression program changes resulting in nanotoxicity. With this approach, it is possible to construct a data set of genomic regions that could be useful for defining toxicity in a manner that is more comprehensive than what is possible with the present toxicology assays.

CCRL2 Expression by Specialized Lung Capillary Endothelial Cells Controls NK-cell Homing in Lung Cancer.

Patterns of receptors for chemotactic factors regulate the homing of leukocytes to tissues. Here we report that the CCRL2/chemerin/CMKLR1 axis represents a selective pathway for the homing of natural killer (NK) cells to the lung. C-C motif chemokine receptor-like 2 (CCRL2) is a nonsignaling seven-transmembrane domain receptor able to control lung tumor growth. CCRL2 constitutive or conditional endothelial cell targeted ablation, or deletion of its ligand chemerin, were found to promote tumor progression in a Kras/p53Flox lung cancer cell model. This phenotype was dependent on the reduced recruitment of CD27- CD11b+ mature NK cells. Other chemotactic receptors identified in lung-infiltrating NK cells by single-cell RNA sequencing (scRNA-seq), such as Cxcr3, Cx3cr1, and S1pr5, were found to be dispensable in the regulation of NK-cell infiltration of the lung and lung tumor growth. scRNA-seq identified CCRL2 as the hallmark of general alveolar lung capillary endothelial cells. CCRL2 expression was epigenetically regulated in lung endothelium and it was upregulated by the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). In vivo administration of low doses of 5-Aza induced CCRL2 upregulation, increased recruitment of NK cells, and reduced lung tumor growth. These results identify CCRL2 as an NK-cell lung homing molecule that has the potential to be exploited to promote NK cell-mediated lung immune surveillance.

The Yin and Yang of epigenetics in the field of nanoparticles.

Nanoparticles (NPs) have become a very exciting research avenue, with multitudinous applications in various fields, including the biomedical one, whereby they have been gaining considerable interest as drug carriers able to increase bioavailability, therapeutic efficiency and specificity of drugs. Epigenetics, a complex network of molecular mechanisms involved in gene expression regulation, play a key role in mediating the effect of environmental factors on organisms and in the etiology of several diseases (e.g., cancers, neurological disorders and cardiovascular diseases). For many of these diseases, epigenetic therapies have been proposed, whose application is however limited by the toxicity of epigenetic drugs. In this review, we will analyze two aspects of epigenetics in the field of NPs: the first is the role that epigenetics play in mediating nanotoxicity, and the second is the possibility of using NPs for delivery of "epi-drugs" to overcome their limitations. We aim to stimulate discussion among specialists, specifically on the potential contribution of epigenetics to the field of NPs, and to inspire newcomers to this exciting technology.

Copper oxide nanoparticles trigger macrophage cell death with misfolding of Cu/Zn superoxide dismutase 1 (SOD1).

BACKGROUND: Copper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity in macrophages exposed to CuO NPs versus copper chloride (CuCl2). METHODS: The mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay. RESULTS: We observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death. CONCLUSIONS: CuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.

Impact on NK cell functions of acute versus chronic exposure to extracellular vesicle-associated MICA: Dual role in cancer immunosurveillance.

Natural killer (NK) cells are innate cytotoxic lymphocytes that play a key role in cancer immunosurveillance thanks to their ability to recognize and kill cancer cells. NKG2D is an activating receptor that binds to MIC and ULBP molecules typically induced on damaged, transformed or infected cells. The release of NKG2D ligands (NKG2DLs) in the extracellular milieu through protease-mediated cleavage or by extracellular vesicle (EV) secretion allows cancer cells to evade NKG2D-mediated immunosurveillance. In this work, we investigated the immunomodulatory properties of the NKG2D ligand MICA*008 associated to distinct populations of EVs (i.e., small extracellular vesicles [sEVs] and medium size extracellular vesicles [mEVs]). By using as model a human MICA*008-transfected multiple myeloma (MM) cell line, we found that this ligand is present on both vesicle populations. Interestingly, our findings reveal that NKG2D is specifically involved in the uptake of vesicles expressing its cognate ligand. We provide evidence that MICA*008-expressing sEVs and mEVs are able on one hand to activate NK cells but, following prolonged stimulation induce a sustained NKG2D downmodulation leading to impaired NKG2D-mediated functions. Moreover, our findings show that MICA*008 can be transferred by vesicles to NK cells causing fratricide. Focusing on MM as a clinically and biologically relevant model of tumour-NK cell interactions, we found enrichment of EVs expressing MICA in the bone marrow of a cohort of patients. All together our results suggest that the accumulation of NKG2D ligands associated to vesicles in the tumour microenvironment could favour the suppression of NK cell activity either by NKG2D down-modulation or by fratricide of NK cell dressed with EV-derived NKG2D ligands.

Enriched Environment Cues Suggest a New Strategy to Counteract Glioma: Engineered rAAV2-IL-15 Microglia Modulate the Tumor Microenvironment.

Several types of cancer grow differently depending on the environmental stimuli they receive. In glioma, exposure to an enriched environment (EE) increases the overall survival rate of tumor-bearing mice, acting on the cells that participate to define the tumor microenvironment. In particular, environmental cues increase the microglial production of interleukin (IL)-15 which promotes a pro-inflammatory (antitumor) phenotype of microglia and the cytotoxic activity of natural killer (NK) cells, counteracting glioma growth, thus representing a virtuous mechanism of interaction between NK cells and microglia. To mimic the effect of EE on glioma, we investigated the potential of creating engineered microglia as the source of IL-15 in glioma. We demonstrated that microglia modified with recombinant adeno-associated virus serotype 2 (rAAV2) carrying IL-15 (rAAV2-IL-15), to force the production of IL-15, are able to increase the NK cells viability in coculture. Furthermore, the intranasal delivery of rAAV2-IL-15 microglia triggered the interplay with NK cells in vivo, enhancing NK cell recruitment and pro-inflammatory microglial phenotype in tumor mass of glioma-bearing mice, and ultimately counteracted tumor growth. This approach has a high potential for clinical translatability, highlighting the therapeutic efficacy of forced IL-15 production in microglia: the delivery of engineered rAAV2-IL-15 microglia to boost the immune response paves the way to design a new perspective therapy for glioma patients.

Histone-deacetylase 8 drives the immune response and the growth of glioma.

Many epigenetic modifications occur in glioma, in particular the histone-deacetylase class proteins play a pivotal role in glioma development, driving the proliferation rate and the invasiveness of tumor cells, and modulating the tumor microenvironment. In this study, we evaluated the role of the histone deacetylase HDAC8 in the regulation of the immune response in glioma and tumor growth. We found that inhibition of HDAC8 by the specific inhibitor PCI-34051 reduces tumor volume in glioma mouse models. We reported that HDAC8 modulates the viability and the migration of human and murine glioma cells. Interestingly, HDAC8 inhibition increases the acetylation of alpha-tubulin, suggesting this epigenetic modification controls glioma migration. Furthermore, we identify HDAC8 as a key molecule that supports a poorly immunogenic tumor microenvironment, modulating microglial phenotype and regulating the gene transcription of NKG2D ligands that trigger the Natural Killer cell-mediated cytotoxicity of tumor cells. Altogether, these results identify HDAC8 as a key actor in glioma growth and tumor microenvironment, and pave the way to a better knowledge of the molecular mechanisms of immune escape in glioma.

NK cell surveillance of hematological malignancies. Therapeutic implications and regulation by chemokine receptors.

NK cells are circulating innate lymphoid cells that constantly move from bloodstream into tissues, exerting several functions including tumor surveillance. For this reason, NK cells are considered attractive target for cancer immunotherapy. Several strategies are employed to harness NK cell efficacy especially in hematological tumors, including adoptive transfer, genetic manipulation to overexpress chimeric antigen receptors and cytokine or immunomodulatory drug treatments of ex-vivo cultivated and expanded NK cells. Several chemokine receptors support NK cell tissue homing and are required for efficient tumor infiltration. Nevertheless, chemokine receptor expression is often insufficient, or their respective ligands may not be expressed in the tumor microenvironment, thus limiting NK cell localization at the tumor site. Therefore, strategies to implement expression or promote the function of the correct chemokine receptor/ligand axes have been employed in the last years with promising results in preclinical models. In this review, we discuss how chemokine receptors and their ligands regulate the trafficking and localization of NK cells in hematological tumors and how the chemokine function can be manipulated to improve current therapeutic approaches.

Epigenetics of aging and disease: a brief overview.

Aging is an important risk factor for several human diseases such as cancer, cardiovascular disease and neurodegenerative disorders, resulting from a combination of genetic and environmental factors (e.g., diet, smoking, obesity and stress), which, at molecular level, cause changes in gene expression underlying the decline of physiological function. Epigenetics, which include mechanisms regulating gene expression independently of changes to DNA sequence, regulate gene expression by modulating the structure of chromatin or by regulating the binding of transcriptional machinery to DNA. Several studies showed that an impairment of epigenetic mechanisms promotes alteration of gene expression underlying several aging-related diseases. Alteration of these mechanisms is also linked with changes of gene expression that occurs during aging processes of different tissues. In this review, we will outline the potential role of epigenetics in the onset of two age-related pathologies, cancer and cardiovascular diseases.

NK Cell Anti-Tumor Surveillance in a Myeloid Cell-Shaped Environment.

NK cells are innate lymphoid cells endowed with cytotoxic capacity that play key roles in the immune surveillance of tumors. Increasing evidence indicates that NK cell anti-tumor response is shaped by bidirectional interactions with myeloid cell subsets such as dendritic cells (DCs) and macrophages. DC-NK cell crosstalk in the tumor microenvironment (TME) strongly impacts on the overall NK cell anti-tumor response as DCs can affect NK cell survival and optimal activation while, in turn, NK cells can stimulate DCs survival, maturation and tumor infiltration through the release of soluble factors. Similarly, macrophages can either shape NK cell differentiation and function by expressing activating receptor ligands and/or cytokines, or they can contribute to the establishment of an immune-suppressive microenvironment through the expression and secretion of molecules that ultimately lead to NK cell inhibition. Consequently, the exploitation of NK cell interaction with DCs or macrophages in the tumor context may result in an improvement of efficacy of immunotherapeutic approaches.

Tumor inhibition or tumor promotion? The duplicity of CXCR3 in cancer.

Tumor tissue includes cancer cells and normal stromal cells such as vascular endothelial cells, connective tissue cells (cancer associated fibroblast, mesenchymal stem cell), and immune cells (tumor-infiltrating lymphocytes or TIL, dendritic cells, eosinophils, basophils, mast cells, tumor-associated macrophages or TAM, myeloid-derived suppressor cells or MDSC). Anti-tumor activity is mainly mediated by infiltration of NK cells, Th1 and CD8+ T cells, and correlates with expression of NK cell and T cell attracting chemokines. Nevertheless, cancer cells hijack tissue homeostasis through secretion of cytokines and chemokines that mediate not only the induction of an inflamed status that supports cancer cell survival and growth, but also the recruitment and/or activation of immune suppressive cells. CXCL9, CXCL10, and CXCL11 are known for their tumor-inhibiting properties, but their overexpression in several hematologic and solid tumors correlates with disease severity, suggesting a role in tumor promotion. The dichotomous nature of CXCR3 ligands activity mainly depends on several molecular mechanisms induced by cancer cells themselves able to divert immune responses and to alter the whole local environment. A deep understanding of the nature of such phenomenon may provide a rationale to build up a CXCR3/ligand axis targeting strategy. In this review, we will discuss the role of CXCR3 in cancer progression and in regulation of anti-tumor immune response and immunotherapy.

Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles.

Iron nanoparticles (NPs) have been proposed as a tool in very different fields such as environmental remediation and biomedical applications, including food fortification against iron deficiency, even if there is still concern about their safety. Here, we propose Xenopus laevis embryos as a suitable model to investigate the toxicity and the bio-interactions at the intestinal barrier of Fe3O4 and zerovalent iron (ZVI) NPs compared to Fe(II) and (III) salts in the 5 to 100 mg Fe/L concentration range using the Frog Embryo Teratogenesis Assay in Xenopus (FETAX). Our results demonstrated that, at concentrations at which iron salts induce adverse effects, both iron NPs do not cause acute toxicity or teratogenicity even if they accumulate massively in the embryo gut. Prussian blue staining, confocal and electron microscopy allowed mapping of iron NPs in enterocytes, along the paracellular spaces and at the level of the basement membrane of a well-preserved intestinal epithelium. Furthermore, the high bioaccumulation factor and the increase in embryo length after exposure to iron NPs suggest greater iron intake, an essential element for organisms. Together, these results improve the knowledge on the safety of orally ingested iron NPs and their interaction with the intestinal barrier, useful for defining the potential risks associated with their use in food/feed fortification.

Targeting of CXCR3 improves anti-myeloma efficacy of adoptively transferred activated natural killer cells.

BACKGROUND: The peculiar multiple myeloma microenvironment, characterized by up-regulated levels of several inflammatory chemokines, including the CXCR3 receptor ligands CXCL9 and CXCL10, limits NK cell positioning into the bone marrow by interfering with CXCR4 function. It is still unclear if the consequent reduced influx of transferred cells into the tumor represents a potential limiting factor for the success of NK cell-based adoptive therapy. We hypothesize that inhibition of CXCR3 function on NK cells will result in increased tumor clearance, due to higher NK cell bone marrow infiltration. METHODS: Since different activation protocols differently affect expression and function of homing receptors, we analyzed the bone marrow homing properties and anti-tumor efficacy of NK cells stimulated in vitro with two independent protocols. NK cells were purified from wild-type or Cxcr3-/- mice and incubated with IL-15 alone or with a combination of IL-12, IL-15, IL-18 (IL-12/15/18). Alternatively, CXCR3 function was neutralized in vivo using a specific blocking antibody. NK cell functional behavior and tumor growth were analyzed in bone marrow samples by FACS analysis. RESULTS: Both activation protocols promoted degranulation and IFN-γ production by donor NK cells infiltrating the bone marrow of tumor-bearing mice, although IL-15 promoted a faster but more transient acquisition of functional capacities. In addition, IL-15-activated cells accumulated more in the bone marrow in a short time but showed lower persistence in vivo. Targeting of CXCR3 increased the bone marrow homing capacity of IL-15 but not IL12/15/18 activated NK cells. This effect correlated with a superior and durable myeloma clearance capacity of transferred cells in vivo. CONCLUSIONS: Our results demonstrate that in vitro activation affects NK cell anti-myeloma activity in vivo by regulating their BM infiltration. Furthermore, we provided direct evidence that CXCR3 restrains NK cell anti-tumor capacity in vivo according to the activation protocol used, and that the effects of NK cell-based adoptive immunotherapy for multiple myeloma can be improved by increasing their bone marrow homing through CXCR3 inhibition.

The Atypical Receptor CCRL2 Is Essential for Lung Cancer Immune Surveillance.

CCRL2 is a nonsignaling seven-transmembrane domain receptor. CCRL2 binds chemerin, a protein that promotes chemotaxis of leukocytes, including macrophages and natural killer (NK) cells. In addition, CCRL2 controls the inflammatory response in different pathologic settings, such as hypersensitivity, inflammatory arthritis, and experimental autoimmune encephalitis. Here, we investigated the role of CCRL2 in the regulation of lung cancer-related inflammation. The genetic deletion of Ccrl2 promoted tumor progression in urethane-induced and in Kras G12D/+/p53 LoxP lung tumor mouse models. Similarly, a Kras-mutant lung tumor displayed enhanced growth in Ccrl2-deficient mice. This phenotype was associated with a reduced inflammatory infiltrate characterized by the impaired recruitment of several leukocyte populations including NK cells. Bone marrow chimeras showed that CCRL2 expression by the nonhematopoietic cell compartment was responsible for the increased tumor formation observed in Kras-mutant Ccrl2-deficient mice. In human and mouse lungs, CCRL2 was expressed by a fraction of CD31+ endothelial cells, where it could control NK infiltration. Elevated CCRL2 expression in biopsies from human lung adenocarcinoma positively correlated with clinical outcome. These results provide evidence for a crucial role of CCRL2 in shaping an anti-lung tumor immune response.