Modulatory Effects of Heat-Inactivated Streptococcus Thermophilus Strain 7 on the Inflammatory Response: A Study on an Animal Model with TLR3-Induced Intestinal Injury.

Rotavirus infections result in severe gastroenteritis with a detrimental inflammatory response in the intestine. Because probiotics have an anti-inflammatory effect and can modulate the gut microbiota profile, they can be used as a biotherapy for inflammatory intestinal diseases. In this study, we isolated Streptococcus thermophilus strain 7 (ST7) from cow milk and examined the effect of heat-inactivated ST7 on the intestinal histopathological score, inflammatory cytokine levels, T-cell activation and effector function, and microbiome profile in a mouse model with intestinal injury induced by polyinosinic-polycytidylic acid (poly I:C), a Toll-like receptor 3 agonist. The results indicated that ST7 treatment prevented weight loss and intestinal injury and prevented the upregulation of serum interleukin-6 (IL-6), tumor necrosis factor-α, and IL-15 levels in intestinal epithelial cells; prevented the upregulation of inflammation-associated Gammaproteobacteria and Alistipes; and increased the levels of Firmicutes in fecal microbiota after poly I:C stimulation. ST7 treatment also increased the serum interferon-γ (IFN-γ) level and promoted the expression of IFN-γ in both CD8 and CD4 T cells. In summary, ST7 prevented the inflammatory response, promoted the T-cell effector function, and modulated the microbiota profile of mice with poly I:C-induced small intestine injury.

KI Essence extract (a spleen-tonifying formula) promotes neurite outgrowth, alleviates oxidative stress and hypomyelination, and modulates microbiome in maternal immune activation offspring.

Mushrooms and Chinese traditional herbs have bioactive nutraceuticals with multiple therapeutic functions, including antioxidant and antibacterial activities and microbiome modulation properties. Mushroom-derived bioactive compounds are used in medicines for the treatment of neurological disorders with abnormal brain-gut-microbiome axis. This study examined the effects of KI Essence extract, a spleen-tonifying formula, on neurite growth, antioxidant activity, hypomyelination modulation, and the microbiome profile in lipopolysaccharide (LPS)-induced maternal immune activation (MIA) offspring. The KI Essence extract induced PC12 cell neurite growth by increasing extracellular signal-regulated kinase (ERK) phosphorylation, promoting 2,2'-diphenyl-1-picrylhydrazyl radical scavenging activity, reducing the level of tert-butylhydroperoxide-induced lipid peroxidation in brain homogenates, protecting PC12 cells from H2O2-induced cell death (through the inhibition of ERK phosphorylation), alleviating hypomyelination, and downregulating interleukin-1ß through LPS-activated microglia production; moreover, the numbers of Enterobacteriaceae, Actinobacteria, Peptostreptococcaceae, Erysipelotrichaceae, and Bifidobacterium bacteria in MIA offspring increased. In summary, the KI Essence extract promotes neurite outgrowth, alleviates oxidative stress and hypomyelination, and modulates microbiota dysbiosis in MIA offspring.

Anti-Angiogenetic and Anti-Lymphangiogenic Effects of a Novel 2-Aminobenzimidazole Derivative, MFB.

Background and Purpose: Benzimidazoles have attracted much attention over the last few decades due to their broad-spectrum pharmacological properties. Increasing evidence is showing the potential use of benzimidazoles as anti-angiogenic agents, although the mechanisms that impact angiogenesis remain to be fully defined. In this study, we aim to investigate the anti-angiogenic mechanisms of MFB, a novel 2-aminobenzimidazole derivative, to develop a novel angiogenesis inhibitor. Experimental Approach: MTT, BrdU, migration and invasion assays, and immunoblotting were employed to examine MFB's effects on vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, migration, invasion, as well as signaling molecules activation. The anti-angiogenic effects of MFB were analyzed by tube formation, aorta ring sprouting, and matrigel plug assays. We also used a mouse model of lung metastasis to determine the MFB's anti-metastatic effects. Key Results: MFB suppressed cell proliferation, migration, invasion, and endothelial tube formation of VEGF-A-stimulated human umbilical vascular endothelial cells (HUVECs) or VEGF-C-stimulated lymphatic endothelial cells (LECs). MFB suppressed VEGF-A and VEGF-C signaling in HUVECs or LECs. In addition, MFB reduced VEGF-A- or tumor cells-induced neovascularization in vivo. MFB also diminished B16F10 melanoma lung metastasis. The molecular docking results further showed that MFB may bind to VEGFR-2 rather than VEGF-A with high affinity. Conclusions and Implications: These observations indicated that MFB may target VEGF/VEGFR signaling to suppress angiogenesis and lymphangiogenesis. It also supports the role of MFB as a potential lead in developing novel agents for the treatment of angiogenesis- or lymphangiogenesis-associated diseases and cancer.

Identification of Differentially Expressed Genes in Different Glioblastoma Regions and Their Association with Cancer Stem Cell Development and Temozolomide Response.

The molecular heterogeneity of gene expression profiles of glioblastoma multiforme (GBM) are the most important prognostic factors for tumor recurrence and drug resistance. Thus, the aim of this study was to identify potential target genes related to temozolomide (TMZ) resistance and GBM recurrence. The genomic data of patients with GBM from The Cancer Genome Atlas (TCGA; 154 primary and 13 recurrent tumors) and a local cohort (29 primary and 4 recurrent tumors), samples from different tumor regions from a local cohort (29 tumor and 25 peritumoral regions), and Gene Expression Omnibus data (GSE84465, single-cell RNA sequencing; 3589 cells) were included in this study. Critical gene signatures were identified based an analysis of differentially expressed genes (DEGs). DEGs were further used to evaluate gene enrichment levels among primary and recurrent GBMs and different tumor regions through gene set enrichment analysis. Protein-protein interactions (PPIs) were incorporated into gene regulatory networks to identify the affected metabolic pathways. The enrichment levels of 135 genes were identified in the peritumoral regions as being risk signatures for tumor recurrence. Fourteen genes (DVL1, PRKACB, ARRB1, APC, MAPK9, CAMK2A, PRKCB, CACNA1A, ERBB4, RASGRF1, NF1, RPS6KA2, MAPK8IP2, and PPM1A) derived from the PPI network of 135 genes were upregulated and involved in the regulation of cancer stem cell (CSC) development and relevant signaling pathways (Notch, Hedgehog, Wnt, and MAPK). The single-cell data analysis results indicated that 14 key genes were mainly expressed in oligodendrocyte progenitor cells, which could produce a CSC niche in the peritumoral region. The enrichment levels of 336 genes were identified as biomarkers for evaluating TMZ resistance in the solid tumor region. Eleven genes (ARID5A, CDC42EP3, CDKN1A, FLT3, JUNB, MAP2K3, MYBPC2, RGS14, RNASEK, TBC1D30, and TXNDC11) derived from the PPI network of 336 genes were upregulated and may be associated with a high risk of TMZ resistance; these genes were identified in both the TCGA and local cohorts. Furthermore, the expression patterns of ARID5A, CDKN1A, and MAP2K3 were identical to the gene signatures of TMZ-resistant cell lines. The identified enrichment levels of the two gene sets expressed in tumor and peritumoral regions are potentially helpful for evaluating TMZ resistance in GBM. Moreover, these key genes could be used as biomarkers, potentially providing new molecular strategies for GBM treatment.

Maternal Immune Activation Causes Social Behavior Deficits and Hypomyelination in Male Rat Offspring with an Autism-Like Microbiota Profile.

Maternal immune activation (MIA) increases the risk of autism spectrum disorder (ASD) in offspring. Microbial dysbiosis is associated with ASD symptoms. However, the alterations in the brain-gut-microbiota axis in lipopolysaccharide (LPS)-induced MIA offspring remain unclear. Here, we examined the social behavior, anxiety-like and repetitive behavior, microbiota profile, and myelination levels in LPS-induced MIA rat offspring. Compared with control offspring, MIA male rat offspring spent less time in an active social interaction with stranger rats, displayed more anxiety-like and repetitive behavior, and had more hypomyelination in the prefrontal cortex and thalamic nucleus. A fecal microbiota analysis revealed that MIA offspring had a higher abundance of Alistipes, Fusobacterium, and Ruminococcus and a lower abundance of Coprococcus, Erysipelotrichaies, and Actinobacteria than control offspring, which is consistent with that of humans with ASD. The least absolute shrinkage and selection operator (LASSO) method was applied to determine the relative importance of the microbiota, which indicated that the abundance of Alistipes and Actinobacteria was the most relevant for the profile of defective social behavior, whereas Fusobacterium and Coprococcus was associated with anxiety-like and repetitive behavior. In summary, LPS-induced MIA offspring showed an abnormal brain-gut-microbiota axis with social behavior deficits, anxiety-like and repetitive behavior, hypomyelination, and an ASD-like microbiota profile.

Detection of PD-L1 Expression in Temozolomide-Resistant Glioblastoma by Using PD-L1 Antibodies Conjugated with Lipid­Coated Superparamagnetic Iron Oxide.

PURPOSE: Targeted superparamagnetic iron oxide (SPIO) nanoparticles are a promising tool for molecular magnetic resonance imaging (MRI) diagnosis. Lipid-coated SPIO nanoparticles have a nonfouling property that can reduce nonspecific binding to off-target cells and prevent agglomeration, making them suitable contrast agents for molecular MRI diagnosis. PD-L1 is a poor prognostic factor for patients with glioblastoma. Most recurrent glioblastomas are temozolomide resistant. Diagnostic probes targeting PD-L1 could facilitate early diagnosis and be used to predict responses to targeted PD-L1 immunotherapy in patients with primary or recurrent glioblastoma. We conjugated lipid-coated SPIO nanoparticles with PD-L1 antibodies to identify PD-L1 expression in glioblastoma or temozolomide-resistant glioblastoma by using MRI. METHODS: The synthesized PD-L1 antibody-conjugated SPIO (PDL1-SPIO) nanoparticles were characterized using dynamic light scattering, zeta potential assays, transmission electron microscopy images, Prussian blue assay, in vitro cell affinity assay, and animal MRI analysis. RESULTS: PDL1-SPIO exhibited a specific binding capacity to PD-L1 of the mouse glioblastoma cell line (GL261). The presence and quantity of PDL1-SPIO in temozolomide-resistant glioblastoma cells and tumor tissue were confirmed through Prussian blue staining and in vivo T2* map MRI, respectively. CONCLUSION: This is the first study to demonstrate that PDL1-SPIO can specifically target temozolomide-resistant glioblastoma with PD-L1 expression in the brain and can be quantified through MRI analysis, thus making it suitable for the diagnosis of PD-L1 expression in temozolomide-resistant glioblastoma in vivo.

A Novel Multiprotein Bridging Factor 1-Like Protein Induces Cyst Wall Protein Gene Expression and Cyst Differentiation in Giardia lamblia.

The capacity to synthesize a protective cyst wall is critical for infectivity of Giardia lamblia. It is of interest to know the mechanism of coordinated synthesis of three cyst wall proteins (CWPs) during encystation, a differentiation process. Multiprotein bridging factor 1 (MBF1) gene family is a group of transcription coactivators that bridge various transcription factors. They are involved in cell growth and differentiation in yeast and animals, or in stress response in fungi and plants. We asked whether Giardia has MBF1-like genes and whether their products influence gene expression. BLAST searches of the Giardia genome database identified one gene encoding a putative MBF1 protein with a helix-turn-helix domain. We found that it can specifically bind to the AT-rich initiator promoters of the encystation-induced cwp1-3 and myb2 genes. MBF1 localized to cell nuclei and cytoplasm with higher expression during encystation. In addition, overexpression of MBF1 induced cwp1-3 and myb2 gene expression and cyst generation. Mutation of the helixes in the helix-turn-helix domain reduced cwp1-3 and myb2 gene expression and cyst generation. Chromatin immunoprecipitation assays confirmed the binding of MBF1 to the promoters with its binding sites in vivo. We also found that MBF1 can interact with E2F1, Pax2, WRKY, and Myb2 transcription factors that coordinately up-regulate the cwp genes during encystation. Using a CRISPR/Cas9 system for targeted disruption of mbf1 gene, we found a downregulation of cwp1-3 and myb2 genes and decrease of cyst generation. Our results suggest that MBF1 is functionally conserved and positively regulates Giardia cyst differentiation.

CD8+CD122+ T cell homeostasis is controlled by different levels of IL-15 trans-presentation.

The homeostasis of CD8+CD122+ T cell requires IL-15 trans-presentation. We use Il15ra mutant mice and bone marrow chimeras to assess the role of IL-15 trans-presentation level in CD8+CD122+ T cells homeostasis. We demonstrate that CD8+CD122+ T cells require different levels of IL-15 trans-presentation to support their homeostasis.

Chinese herbal medicine SS-1 inhibits T cell activation and abrogates TH responses in Sjögren's syndrome.

BACKGROUND/PURPOSE: Sjögren's syndrome (SS) is an autoimmune disease and its conventional treatment has exhibited limited therapeutic efficacy. Traditional Chinese medicine has been demonstrated to ameliorate the sicca symptoms of SS by decreasing the level of TH1 and TH2 cytokines and increasing salivary flow rate. A newly designed traditional Chinese medicine, SS-1, showed improved efficacy in alleviating the dryness symptoms of SS patients in the National Taiwan SS cohort investigation. Here, we investigated the effect of SS-1 on T cell responses. METHODS: SS-1 was authenticated and its major compounds were verified by high-performance liquid chromatography. We examined the effects of SS-1 on the activation and TH1, TH2, and TH17 polarization of murine T cells. We also determined the level of TH1, TH2, and TH17 cytokine RNA in peripheral blood mononuclear cells of SS patients before and after SS-1 treatment. RESULTS: SS-1 treatment inhibits the activation and TH1, TH2, and IL-17A+IFNγ+ TH polarization of murine T cells. SS-1 treatment also significantly reduces IFN-γ, IL-4, and IL-13 expression, and moderately reduces IL-17A expression in peripheral blood mononuclear cells of SS patients. CONCLUSION: Our results suggest that SS-1 inhibits T cell activation and diminishes TH1, TH2, and IL-17+IFN-γ+ TH responses in SS patients.

Radiomic Immunophenotyping of GSEA-Assessed Immunophenotypes of Glioblastoma and Its Implications for Prognosis: A Feasibility Study.

Characterization of immunophenotypes in glioblastoma (GBM) is important for therapeutic stratification and helps predict treatment response and prognosis. Radiomics can be used to predict molecular subtypes and gene expression levels. However, whether radiomics aids immunophenotyping prediction is still unknown. In this study, to classify immunophenotypes in patients with GBM, we developed machine learning-based magnetic resonance (MR) radiomic models to evaluate the enrichment levels of four immune subsets: Cytotoxic T lymphocytes (CTLs), activated dendritic cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs). Independent testing data and the leave-one-out cross-validation method were used to evaluate model effectiveness and model performance, respectively. We identified five immunophenotypes (G1 to G5) based on the enrichment level for the four immune subsets. G2 had the worst prognosis and comprised highly enriched MDSCs and lowly enriched CTLs. G3 had the best prognosis and comprised lowly enriched MDSCs and Tregs and highly enriched CTLs. The average accuracy of T1-weighted contrasted MR radiomics models of the enrichment level for the four immune subsets reached 79% and predicted G2, G3, and the "immune-cold" phenotype (G1) according to our radiomics models. Our radiomic immunophenotyping models feasibly characterize the immunophenotypes of GBM and can predict patient prognosis.

DNA topoisomerase IIIß promotes cyst generation by inducing cyst wall protein gene expression in Giardia lamblia.

Giardia lamblia causes waterborne diarrhoea by transmission of infective cysts. Three cyst wall proteins are highly expressed in a concerted manner during encystation of trophozoites into cysts. However, their gene regulatory mechanism is still largely unknown. DNA topoisomerases control topological homeostasis of genomic DNA during replication, transcription and chromosome segregation. They are involved in a variety of cellular processes including cell cycle, cell proliferation and differentiation, so they may be valuable drug targets. Giardia lamblia possesses a type IA DNA topoisomerase (TOP3ß) with similarity to the mammalian topoisomerase IIIß. We found that TOP3ß was upregulated during encystation and it possessed DNA-binding and cleavage activity. TOP3ß can bind to the cwp promoters in vivo using norfloxacin-mediated topoisomerase immunoprecipitation assays. We also found TOP3ß can interact with MYB2, a transcription factor involved in the coordinate expression of cwp1-3 genes during encystation. Interestingly, overexpression of TOP3ß increased expression of cwp1-3 and myb2 genes and cyst formation. Microarray analysis confirmed upregulation of cwp1-3 and myb2 genes by TOP3ß. Mutation of the catalytically important Tyr residue, deletion of C-terminal zinc ribbon domain or further deletion of partial catalytic core domain reduced the levels of cleavage activity, cwp1-3 and myb2 gene expression, and cyst formation. Interestingly, some of these mutant proteins were mis-localized to cytoplasm. Using a CRISPR/Cas9 system for targeted disruption of top3ß gene, we found a significant decrease in cwp1-3 and myb2 gene expression and cyst number. Our results suggest that TOP3ß may be functionally conserved, and involved in inducing Giardia cyst formation.

Identification of potential biomarkers related to glioma survival by gene expression profile analysis.

BACKGROUND: Recent studies have proposed several gene signatures as biomarkers for different grades of gliomas from various perspectives. However, most of these genes can only be used appropriately for patients with specific grades of gliomas. METHODS: In this study, we aimed to identify survival-relevant genes shared between glioblastoma multiforme (GBM) and lower-grade glioma (LGG), which could be used as potential biomarkers to classify patients into different risk groups. Cox proportional hazard regression model (Cox model) was used to extract relative genes, and effectiveness of genes was estimated against random forest regression. Finally, risk models were constructed with logistic regression. RESULTS: We identified 104 key genes that were shared between GBM and LGG, which could be significantly correlated with patients' survival based on next-generation sequencing data obtained from The Cancer Genome Atlas for gene expression analysis. The effectiveness of these genes in the survival prediction of GBM and LGG was evaluated, and the average receiver operating characteristic curve (ROC) area under the curve values ranged from 0.7 to 0.8. Gene set enrichment analysis revealed that these genes were involved in eight significant pathways and 23 molecular functions. Moreover, the expressions of ten (CTSZ, EFEMP2, ITGA5, KDELR2, MDK, MICALL2, MAP 2 K3, PLAUR, SERPINE1, and SOCS3) of these genes were significantly higher in GBM than in LGG, and comparing their expression levels to those of the proposed control genes (TBP, IPO8, and SDHA) could have the potential capability to classify patients into high- and low- risk groups, which differ significantly in the overall survival. Signatures of candidate genes were validated, by multiple microarray datasets from Gene Expression Omnibus, to increase the robustness of using these potential prognostic factors. In both the GBM and LGG cohort study, most of the patients in the high-risk group had the IDH1 wild-type gene, and those in the low-risk group had IDH1 mutations. Moreover, most of the high-risk patients with LGG possessed a 1p/19q-noncodeletion. CONCLUSION: In this study, we identified survival relevant genes which were shared between GBM and LGG, and those enabled to classify patients into high- and low-risk groups based on expression level analysis. Both the risk groups could be correlated with the well-known genetic variants, thus suggesting their potential prognostic value in clinical application.

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

Interleukin 15 blockade protects the brain from cerebral ischemia-reperfusion injury.

Acute ischemic stroke is followed by a complex interplay between the brain and the immune system in which ischemia-reperfusion leads to a detrimental inflammatory response that causes brain injury. In the brain, IL-15 is expressed by astrocytes, neurons and microglia. Previous study showed that ischemia-reperfusion induces expression of IL-15 by astrocytes. Transgenic over-expression of IL-15 in astrocytes aggravates ischemia-reperfusion brain damage by increasing the levels and promoting the effector functions of CD8+ T and NK cells. Treatment of neonatal rats with IL-15 neutralizing antibody before hypoxia-ischemia induction reduces the infarct volume. However, as stroke-induced inflammatory responses differ between neonate and adult brain, the effects of IL-15 blockade on the injury and immune response arising from stroke in adult animals has remained unclear. In this study, we examined the effect of post-ischemia/reperfusion IL-15 blockade on the pathophysiology of cerebral ischemia-reperfusion in adult mice. Using a cerebral ischemia-reperfusion model, we compared infarct size and the infiltrating immune cells in the brain of wild type (WT) mice and Il15-/- mice lacking NK and memory CD8+ T cells. We also evaluated the effects of IL-15 neutralizing antibody treatment on brain infarct volume, motor function, and the status of brain-infiltrating immune cells in WT mice. Il15-/- mice show a smaller infarct volume and lower numbers of activated brain-infiltrating NK, CD8+ T, and CD4+ T cells compared to WT mice after cerebral ischemia-reperfusion. Post-ischemia/reperfusion IL-15 blockade reduces infarct size and improves motor and locomotor activity. Furthermore, IL-15 blockade reduces the effector function of NK, CD8+ T, and CD4+ T cells in the ischemia-reperfusion brain of WT mice. Ablation of IL-15 responses after cerebral ischemia-reperfusion ameliorates brain injury in adult mice. Therefore, targeting IL-15 is a potential effective therapy for ischemic stroke.

Identification of epidermal growth factor receptor-positive glioblastoma using lipid-encapsulated targeted superparamagnetic iron oxide nanoparticles in vitro.

BACKGROUND: Targeted superparamagnetic iron oxide (SPIO) nanoparticles have emerged as a promising biomarker detection tool for molecular magnetic resonance (MR) image diagnosis. To identify patients who could benefit from Epidermal growth factor receptor (EGFR)-targeted therapies, we introduce lipid-encapsulated SPIO nanoparticles and hypothesized that anti-EGFR antibody cetuximab conjugated of such nanoparticles can be used to identify EGFR-positive glioblastomas in non-invasive T2 MR image assays. The newly introduced lipid-coated SPIOs, which imitate biological cell surface and thus inherited innate nonfouling property, were utilized to reduce nonspecific binding to off-targeted cells and prevent agglomeration that commonly occurs in nanoparticles. RESULTS: The synthesized targeted EGFR-antibody-conjugated SPIO (EGFR-SPIO) nanoparticles were characterized using dynamic light scattering, zeta potential assays, gel electrophoresis mobility shift assays, transmission electron microscopy (TEM) images, and cell line affinity assays, and the results showed that the conjugation was successful. The targeting efficiency of the synthesized EGFR-SPIO nanoparticles was confirmed through Prussian blue staining and TEM images by using glioblastoma cell lines with high or low EGFR expression levels. The EGFR-SPIO nanoparticles preferentially targeted U-251 cells, which have high EGFR expression, and were internalized by cells in a prolonged incubation condition. Moreover, the T2 MR relaxation time of EGFR-SPIO nanoparticles could be used for successfully identifying glioblastoma cells with elevated EGFR expression in vitro and distinguishing U-251 cells from U-87MG cells, which have low EFGR expression. CONCLUSION: These findings reveal that the lipid-encapsulated EGFR-SPIO nanoparticles can specifically target cells with elevated EGFR expression in the three tested human glioblastoma cell lines. The results of this study can be used for noninvasive molecular MR image diagnosis in the future.

Interleukin 15 activates Akt to protect astrocytes from oxygen glucose deprivation-induced cell death.

Astrocytes play a pivotal role in neuronal survival under the condition of post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. IL-15 supports survival of multiple lymphocyte lineages in the peripheral immune system, but the role of IL-15 in inflammatory disease of the central nervous system is not well defined. Recent research has shown an increase of IL-15-expressing astrocytes in the ischemic brain. Since astrocytes promote neuron survival under cerebral ischemia by buffering excess extracellular glutamate and producing growth factors, recovery of astrocyte function could be of benefit for stroke therapy. Here, we report that IL-15 is the pro-survival cytokine that prevents astrocyte death from oxygen glucose deprivation (OGD)-induced damage. Astrocytes up-regulate expression of the IL-15/IL-15Rα complex under OGD, whereas OGD down-regulates the levels of pSTAT5 and pAkt in astrocytes. IL-15 treatment ameliorates the decline of pAkt, decreases the percentage of annexin V+ cells, inhibits the activation of caspase-3, and activates the Akt pathway to promote astrocyte survival in response to OGD. We further identified that activation of Akt, but not PKCα/ßI, is essential for astrocyte survival under OGD. Taken together, this study reveals the function of IL-15 in astrocyte survival via Akt phosphorylation in response to OGD-induced damage.

Adipocyte IL-15 regulates local and systemic NK cell development.

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.

Different NK cell developmental events require different levels of IL-15 trans-presentation.

NK cell development requires IL-15, which is "trans-presented" to IL-15Rßγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)-derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.