CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.

BACKGROUND: TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 (cluster of differentiation) and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis. METHODS: Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms. RESULTS: Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF (vascular endothelial growth factor)-C-induced AKT and eNOS activation in LEC and attenuated NO (nitric oxide) production. Further, CD47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV (adeno-associated virus) 8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC mice in comparison to controls. CONCLUSIONS: These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Diabetic Endothelial Cell Glycogen Synthase Kinase 3ß Activation Induces VCAM1 Ectodomain Shedding.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1's role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1's release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3ß, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3ß activation in control ECs increased ADAM10/17 and VCAM1. A GSK3ß inhibitor reduced active GSK3ß and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3ß activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3ß inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

MicroRNAs: a crossroad that connects obesity to immunity and aging.

Obesity is characterized by an elevated amount of fat and energy storage in the adipose tissue (AT) and is believed to be the root cause of many metabolic diseases (MDs). Obesity is associated with low-grade chronic inflammation in AT. Like obesity, chronic inflammation and MDs are prevalent in the elderly. The resident immune microenvironment is not only responsible for maintaining AT homeostasis but also plays a crucial role in stemming obesity and related MDs. Mounting evidence suggests that obesity promotes activation in resident T cells and macrophages. Additionally, inflammatory subsets of T cells and macrophages accumulated into the AT in combination with other immune cells maintain low-grade chronic inflammation. microRNAs (miRs) are small non-coding RNAs and a crucial contributing factor in maintaining immune response and obesity in AT. AT resident T cells, macrophages and adipocytes secrete various miRs and communicate with other cells to create a potential effect in metabolic organ crosstalk. AT resident macrophages and T cells-associated miRs have a prominent role in regulating obesity by targeting several signaling pathways. Further, miRs also emerged as important regulators of cellular senescence and aging. To this end, a clear link between miRs and longevity has been demonstrated that implicates their role in regulating lifespan and the aging process. Hence, AT and circulating miRs can be used as diagnostic and therapeutic tools for obesity and related disorders. In this review, we discuss how miRs function as biomarkers and impact obesity, chronic inflammation, and aging.

An endogenous aryl hydrocarbon receptor ligand, ITE, induces regulatory T cells and ameliorates experimental colitis.

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that affects millions of people with high morbidity and health care costs. The precise etiology of IBD is unknown, but clear evidence suggests that intestinal inflammation is caused by an excessive immune response to mucosal antigens. Recent studies have shown that activation of the aryl hydrocarbon receptor (AhR) induces regulatory T cells (Tregs) and suppresses autoimmune diseases. In the current study, we investigated if a nontoxic ligand of AhR, 2-(1' H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), can attenuate dextran sodium sulfate-induced colitis. Our studies demonstrated that in mice that received ITE treatment in vivo, colitis pathogenesis, including a decrease in body weight, was significantly reversed along with the systemic and intestinal inflammatory cytokines. ITE increased the expression of Tregs in spleen, mesenteric lymph nodes (MLNs), and colon lamina propria lymphocytes (cLPL) of mice with colitis when compared with controls. This induction of Tregs was reversed by AhR antagonist treatment in vitro. ITE treatment also increased dendritic cells (CD11c+) and decreased macrophages (F4/80+) from the spleen, MLNs, and cLPL in mice with colitis. ITE also reversed the systemic and intestinal frequency of CD4+ T cells during colitis and suppressed inflammatory cytokines including IFN-γ, TNF-α, IL-17, IL-6, and IL-1 as well as induced IL-10 levels. These findings suggest that ITE attenuates colitis through induction of Tregs and reduction in inflammatory CD4+ T cells and cytokines. Therefore, our work demonstrates that the nontoxic endogenous AhR ligand ITE may serve as a therapeutic modality to treat IBD. NEW & NOTEWORTHY We report the novel finding that activation of the aryl hydrocarbon receptor with the nontoxic ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces regulatory T cells (Tregs) and suppresses inflammatory bowel disease (IBD). Our data suggest that ITE diminishes colitis pathology through induction of Tregs; reduces inflammatory cytokines, inflammation score, and macrophage frequency; and induces DCs resulting in amelioration of colitis. Therefore, nontoxic endogenous ITE promotes the induction of Tregs and may be useful for the treatment of IBD.

Dietary Indoles Suppress Delayed-Type Hypersensitivity by Inducing a Switch from Proinflammatory Th17 Cells to Anti-Inflammatory Regulatory T Cells through Regulation of MicroRNA.

Aryl hydrocarbon receptor (AhR) has been shown to have profound influence on T cell differentiation, and use of distinct AhR ligands has shown that whereas some ligands induce regulatory T cells (Tregs), others induce Th17 cells. In the present study, we tested the ability of dietary AhR ligands (indole-3-carbinol [I3C] and 3,3'-diindolylmethane [DIM]) and an endogenous AhR ligand, 6-formylindolo(3,2-b)carbazole (FICZ), on the differentiation and functions of Tregs and Th17 cells. Treatment of C57BL/6 mice with indoles (I3C or DIM) attenuated delayed-type hypersensitivity (DTH) response to methylated BSA and generation of Th17 cells while promoting Tregs. In contrast, FICZ exacerbated the DTH response and promoted Th17 cells. Indoles decreased the induction of IL-17 but promoted IL-10 and Foxp3 expression. Also, indoles caused reciprocal induction of Tregs and Th17 cells only in wild-type (AhR(+/+)) but not in AhR knockout (AhR(-/-)) mice. Upon analysis of microRNA (miR) profile in draining lymph nodes of mice with DTH, treatment with I3C and DIM decreased the expression of several miRs (miR-31, miR-219, and miR-490) that targeted Foxp3, whereas it increased the expression of miR-495 and miR-1192 that were specific to IL-17. Interestingly, treatment with FICZ had precisely the opposite effects on these miRs. Transfection studies using mature miR mimics of miR-490 and miR-1192 that target Foxp3 and IL-17, respectively, or scrambled miR (mock) or inhibitors confirmed that these miRs specifically targeted Foxp3 and IL-17 genes. Our studies demonstrate, to our knowledge for the first time, that the ability of AhR ligands to regulate the differentiation of Tregs versus Th17 cells may depend on miR signature profile.

Deficiency of KLF4 compromises the lung function in an acute mouse model of allergic asthma.

Asthma is a chronic inflammatory disease of the airways and the mechanisms are not fully understood. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of monocytes, granulocyte and myeloid cells at early stage of differentiation. They possess phenotypic plasticity and regulate airway inflammation. We recently reported that Kruppel-like factor 4 (KLF4) regulates MDSC differentiation into fibrocytes, emerging effectors in chronic inflammation. However, the role of KLF4 in asthma is not known. Thymic stromal lymphopoietin (TSLP) is an epithelial cell-derived cytokine and a key initiator of allergic airway inflammation. Given the fact that TSLP promotes Th2 cytokine production that increases MDSC differentiation into fibrocytes, we postulate that KLF4 regulates asthma in a TSLP-dependent manner. In this study, we utilized a model of allergic asthma with ovalbumin challenge (OVA). We found that upon OVA treatment the wild type mice had increased MDSC infiltration into the lung, up-regulation of KLF4 and TSLP gene expression, and higher levels of Th2 cytokines including IL4 and IL13. Consistently, lack of KLF4 expression in monocytes and lung epithelial cells resulted in decreased TSLP expression and lower levels of Th2 cytokines in mice, and fibrocyte generation was compromised. KLF4 deficiency in these cells also led to decreased airway hyperresponsiveness (AHR), a cardinal feature of asthma, as assessed by whole body plethysmography. Moreover, lung fibrosis as measured by trichome staining was attenuated and the population of CD45 + COL1A1+ fibrocytes was diminished in this setting. Together, our results suggest that KLF4 regulates asthma development in a TSLP- and fibrocyte-dependent manner.

Prolonged exposure of resveratrol induces reactive superoxide species-independent apoptosis in murine prostate cells.

Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species-independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species-independent apoptosis and may act as a therapeutic agent against prostate cancer.

Fatty acid amide hydrolase (FAAH) blockade ameliorates experimental colitis by altering microRNA expression and suppressing inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), which is thought to result from immune-mediated inflammatory disorders, leads to high morbidity and health care cost. Fatty acid amide hydrolase (FAAH) is an enzyme crucially involved in the modulation of intestinal physiology through anandamide (AEA) and other endocannabinoids. Here we examined the effects of an FAAH inhibitor (FAAH-II), on dextran sodium sulphate (DSS)-induced experimental colitis in mice. Treatments with FAAH-II improved overall clinical scores by reversing weight loss and colitis-associated pathogenesis. The frequencies of activated CD4+ T cells in spleens, mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and colon lamina propiria (LP) were reduced by FAAH inhibition. Similarly, the frequencies of macrophages, neutrophils, natural killer (NK), and NKT cells in the PPs and LP of mice with colitis declined after FAAH blockade, as did concentrations of systemic and colon inflammatory cytokines. Microarray analysis showed that 26 miRNAs from MLNs and 217 from PPs had a 1.5-fold greater difference in expression after FAAH inhibition. Among them, 8 miRNAs were determined by reverse-transcription polymerase chain reaction (RT-PCR) analysis to have anti-inflammatory properties. Pathway analysis demonstrated that differentially regulated miRNAs target mRNA associated with inflammation. Thus, FAAH-II ameliorates experimental colitis by reducing not only the number of activated T cells but also the frequency of macrophages, neutrophils, and NK/NKT cell, as well as inflammatory miRNAs and cytokine at effector sites in the colon. These studies demonstrate for the first time that FAAH-II inhibitor may suppress colitis through regulation of pro-inflammatory miRNAs expression.

Critical Role of Mast Cells and Peroxisome Proliferator-Activated Receptor γ in the Induction of Myeloid-Derived Suppressor Cells by Marijuana Cannabidiol In Vivo.

Cannabidiol (CBD) is a natural nonpsychotropic cannabinoid from marijuana (Cannabis sativa) with anti-epileptic and anti-inflammatory properties. Effect of CBD on naive immune system is not precisely understood. In this study, we observed that administering CBD into naive mice triggers robust induction of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the peritoneum, which expressed functional arginase 1, and potently suppressed T cell proliferation ex vivo. Furthermore, CBD-MDSC suppressed LPS-induced acute inflammatory response upon adoptive transfer in vivo. CBD-induced suppressor cells were comprised of CD11b(+)Ly6-G(+)Ly6-C(+) granulocytic and CD11b(+)Ly6-G(-)Ly6-C(+) monocytic subtypes, with monocytic MDSC exhibiting higher T cell-suppressive function. Induction of MDSC by CBD was markedly attenuated in Kit-mutant (Kit(W/W-v)) mast cell-deficient mice. MDSC response was reconstituted upon transfer of wild-type bone marrow-derived mast cells in Kit(W/W-v) mice, suggesting the key role of cKit (CD117) as well as mast cells. Moreover, mast cell activator compound 48/80 induced significant levels of MDSC in vivo. CBD administration in mice induced G-CSF, CXCL1, and M-CSF, but not GM-CSF. G-CSF was found to play a key role in MDSC mobilization inasmuch as neutralizing G-CSF caused a significant decrease in MDSC. Lastly, CBD enhanced the transcriptional activity of peroxisome proliferator-activated receptor γ in luciferase reporter assay, and PPAR-γ selective antagonist completely inhibited MDSC induction in vivo, suggesting its critical role. Together, the results suggest that CBD may induce activation of PPAR-γ in mast cells leading to secretion of G-CSF and consequent MDSC mobilization. CBD being a major component of Cannabis, our study indicates that marijuana may modulate or dysregulate the immune system by mobilizing MDSC.

Inverse correlation of expression of microRNA-140-5p with progression of multiple sclerosis and differentiation of encephalitogenic T helper type 1 cells.

The role of microRNA in the regulation of encephalitogenic T-cell development is of interest in understanding the pathogenesis of multiple sclerosis (MS). Direct binding of microRNAs to their target mRNAs usually suppresses gene expression and facilitates mRNA degradation. In this study, we observed that the expression of several microRNAs was significantly altered in patients with MS. Interestingly, the expression of miR-140-5p, among other microRNAs, was significantly decreased in the peripheral blood mononuclear cells of patients with MS, and this microRNA may regulate encephalitogenic T helper type 1 (Th1) cell differentiation. The expression level of miR-140-5p was inversely correlated with disease severity with greater reduction in relapsing disease compared with remitting disease. Transfection of synthetic miR-140-5p in peripheral blood mononuclear cells suppressed encephalitogenic Th1 differentiation. Signal transducer and activator of transcription 1 (STAT1) was the functional target of miR-140-5p - transfection of the synthetic miR-140-5p suppressed activation of STAT1 and the expression of its downstream target, T-bet. Our results suggested that miR-140-5p is probably involved in the regulation of encephalitogenic T cells in the pathogenesis of MS.

MicroRNA-155 deletion promotes tumorigenesis in the azoxymethane-dextran sulfate sodium model of colon cancer.

Clinical studies have linked microRNA-155 (miR-155) expression in the tumor microenvironment to poor prognosis. However, whether miR-155 upregulation is predictive of a pro- or antitumorigenic response is unclear, as the limited preclinical data available remain controversial. We examined miR-155 expression in tumor tissue from colon cancer patients. Furthermore, we investigated the role of this microRNA in proliferation and apoptosis, inflammatory processes, immune cell populations, and transforming growth factor-ß/SMAD signaling in a chemically induced (azoxymethane-dextran sulfate sodium) mouse model of colitis-associated colon cancer. We found a higher expression of miR-155 in the tumor region than in nontumor colon tissue of patients with colon cancer. Deletion of miR-155 in mice resulted in a greater number of polyps/adenomas, an increased symptom severity score, a higher grade of epithelial dysplasia, and a decrease in survival. Surprisingly, these findings were associated with an increase in apoptosis in the normal mucosa, but there was no change in proliferation. The protumorigenic effects of miR-155 deletion do not appear to be driven solely by dysregulation of inflammation, as both genotypes had relatively similar levels of inflammatory mediators. The enhanced tumorigenic response in miR-155(-/-) mice was associated with alterations in macrophages and neutrophils, as markers for these populations were decreased and increased, respectively. Furthermore, we demonstrated a greater activation of the transforming growth factor-ß/SMAD pathway in miR-155(-/-) mice, which was correlated with the increased tumorigenesis. Given the multiple targets of miR-155, careful evaluation of its role in tumorigenesis is necessary prior to any consideration of its potential as a biomarker and/or therapeutic target in colon cancer.

Weight loss following diet-induced obesity does not alter colon tumorigenesis in the AOM mouse model.

Obesity presents a significant public health concern given its association with increased cancer incidence, unfavorable prognosis, and metastasis. However, there is very little literature on the effects of weight loss, following obesity, on risk for colon cancer or liver cancer. Therefore, we sought to study whether intentional weight loss through diet manipulation was capable of mitigating colon and liver cancer in mice. We fed mice with a high-fat diet (HFD) comprised of 47% carbohydrates, 40% fat, and 13% protein for 20 wk to mimic human obesity. Subsequently, azoxymethane (AOM) was used to promote colon and liver carcinogenesis. A subset of obese mice was then switched to a low-fat diet (LFD) containing 67.5% carbohydrate, 12.2% fat, and 20% protein to promote intentional weight loss. Body weight loss and excess fat reduction did not protect mice from colon cancer progression and liver dysplastic lesion in the AOM-chemical-cancer model even though these mice had improved blood glucose and leptin levels. Intentional weight loss in AOM-treated mice actually produced histological changes that resemble dysplastic alterations in the liver and presented a higher percentage of F4/80+CD206+ macrophages and activated T cells (CD4+CD69+) in the spleen and lymph nodes, respectively. In addition, the liver of AOM-treated mice exposed to a HFD during the entire period of the experiment exhibited a marked increase in proliferation and pNF-κB activation. Altogether, these data suggest that intentional weight loss following chemical-induced carcinogenesis does not affect colon tumorigenesis but may in fact negatively impact liver repair mechanisms.

Chemokine and cytokine levels in inflammatory bowel disease patients.

Crohn's disease (CD) and ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), are chronic, relapsing, and tissue destructive lesions that are accompanied by the uncontrolled activation of effector immune cells in the mucosa. Recent estimates indicate that there are 1.3 million annual cases of IBD in the United States, 50% of which consists of CD and 50% of UC. Chemokines and cytokines play a pivotal role in the regulation of mucosal inflammation by promoting leukocyte migration to sites of inflammation ultimately leading to tissue damage and destruction. In recent years, experimental studies in rodents have led to a better understanding of the role played by these inflammatory mediators in the development and progression of colitis. However, the clinical literature on IBD remains limited. Therefore, the aim of this study was to evaluate systemic concentrations of key chemokines and cytokines in forty-two IBD patients with a range of disease activity compared to levels found in ten healthy donors. We found a significant increase in an array of chemokines including macrophage migration factor (MIF), CCL25, CCL23, CXCL5, CXCL13, CXCL10, CXCL11, MCP1, and CCL21 in IBD patients as compared to normal healthy donors (P<0.05). Further, we also report increases in the inflammatory cytokines IL-16, IFN-γ, IL-1ß and TNF-α in IBD patients when compared to healthy donors (P<0.05). These data clearly indicate an increase in circulating levels of specific chemokines and cytokines that are known to modulate systemic level through immune cells results in affecting local intestinal inflammation and tissue damage in IBD patients. Blockade of these inflammatory mediators should be explored as a mechanism to alleviate or even reverse symptoms of IBD.

Exposure to diethylstilbestrol during pregnancy modulates microRNA expression profile in mothers and fetuses reflecting oncogenic and immunological changes.

Prenatal exposure to diethylstilbestrol (DES) is known to cause an increased susceptibility to a wide array of clinical disorders in humans. Previous studies from our laboratory demonstrated that prenatal exposure to DES induces thymic atrophy and apoptosis in the thymus. In the current study, we investigated if such effects on the thymus result from alterations in the expression of microRNA (miR). To that end, pregnant C57BL/6 mice who were exposed to DES and miR profiles in thymocytes of both the mother and fetuses on postnatal day 3 (gestation day 17) were studied. Of the 609 mouse miRs examined, we noted 59 altered miRs that were common for both mothers and fetuses, whereas 107 altered miRs were specific to mothers only and 101 altered miRs were specific to fetuses only. Upon further analyses in the fetuses, we observed that DES-mediated changes in miR expression may regulate genes involved in important functions, such as apoptosis, autophagy, toxicity, and cancer. Of the miRs that showed decreased expression following DES treatment, miR-18b and miR-23a were found to possess complementary sequences and binding affinity for 3' untranslated regions of the Fas ligand (FasL) and Fas, respectively. Transfection studies confirmed that DES-mediated downregulation of miR-18b and miR-23a led to increased FasL and Fas expression. These data demonstrated that prenatal DES exposure can cause alterations in miRs, leading to changes in the gene expression, specifically, miR-mediated increased expression in FasL and Fas causing apoptosis and thymic atrophy.

Mononuclear iron(iii) complexes of tridentate ligands with efficient nuclease activity and studies of their cytotoxicity.

Mononuclear mono- and bis-chelated iron(iii) complexes [Fe(Phimp)(H2O)(OMe)Cl], 1, [Fe(Phimp)Cl2], 2, [Fe(Me-Phimp)Cl2], 3, [Fe(N-Phimp)Cl2], 4, [Fe(Phimp)2](ClO4), 5, [Fe(Me-Phimp)2](NO3)·H2O, 6·H2O, and [Fe(N-Phimp)2](ClO4), 7, derived from tridentate ligands, have been synthesised and characterized. The X-ray crystal structures of the complexes 2, 4, 5 and 6·H2O were determined. The high-spin iron(iii) complexes were redox active and exhibited the Fe(iii)/Fe(ii) couple. The DNA binding affinity of these complexes was assessed using absorption, fluorescent intercalator displacement assays and circular dichroism spectral studies. Gel electrophoresis studies with DNA and complexes 1, 2, 3 and 6 showed efficient nuclease activity via a hydroxyl radical generated through a Fenton-type reaction mechanism. In situ reactive oxygen species generation has been further supported via DPPH (2,2-diphenyl-1-picrylhydrazine) radical quenching studies as well as theoretical studies. The cytotoxicities of the complexes were determined using the MCF7 cell line; the cytotoxicities (IC50 values) obtained for 1, 2, 3 and 5 were 0.67 ± 0.31, 0.46 ± 0.07, 0.87 ± 0.25 and 1.53 ± 0.41 µM, respectively, and complexes 1-6 were found to be non-toxic to normal HEK cell lines. An acridine orange staining assay for the complexes (1-6) supported cell death, probably via an apoptotic mechanism.

Single Nucleotide Polymorphisms in IL-10, IL-12p40, and IL-13 Genes and Susceptibility to Glioma.

Glioma is one of the most aggressive and most common tumors of the central nervous system (CNS) in humans. The exact causes of glioma are not well known, but evidence suggests the involvement of genetic factors in addition to environmental risk factors. The present study aimed to determine whether polymorphisms in IL-10-1082A/G, IL-12p40 1188C/A, and IL-13+2044G/A (rs20541) are associated with the incidence of glioma in Iraqi patients. Ninety-six patients with different grades of glioma and 40 apparently healthy individuals were recruited. A blood sample and genomic DNA were collected from all subjects. The amplification refractory mutation system and sequence-specific primer polymerase chain reaction (PCR) were used for genotyping of IL-10-1082A/G and IL-12p40 1188C/A, respectively; whereas, the IL-13+2044G/A was detected by DNA sequencing after amplification of the genes by PCR. All SNPs were within Hardy-Weinberg equilibrium and each appeared in three genotypes in patients and controls. In IL-10-1082A/G, these genotypes frequencies were AA (75%), AG (22.93%) and GG (2.07%) in patients as compared to similar frequencies (62.5%), (27.5%) and (10%) respectively, in controls. The variant IL-12p40 1188C/A genotype was AA (72.92%), AC (23.96%), and CC (3.13%%) in patients as compared to 65%, 30%, and 5%, respectively, in controls. The frequencies of IL-13+2044G/A genotypes (GG, GA, and AA) were 89.58%, 9.37%, and 1.04% among patients versus 47.5%, 32.5% and 20%, respectively, among controls. These results suggest a protective role of mutant alleles G and A in IL-10-1082A/G and IL-13+2044G/A against gliomas. Further studies with more rigorous parameter designs will be needed to confirm the current findings.

Distinct microRNA expression profile and targeted biological pathways in functional myeloid-derived suppressor cells induced by Δ9-tetrahydrocannabinol in vivo: regulation of CCAAT/enhancer-binding protein α by microRNA-690.

Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in myeloid expansion and differentiation likely play crucial roles in this process and therefore in cannabinoid-induced immunosuppression.

miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses.

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health-care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR-155, a microRNA, is up-regulated in various inflammatory disease states, including IBD, and is a positive regulator of T-cell responses. To date, no reports have defined a function for miR-155 with regard to cellular responses in IBD. Using an acute experimental colitis model, we found that miR-155(-/-) mice, as compared to wild-type control mice, have decreased clinical scores, a reversal of colitis-associated pathogenesis, and reduced systemic and mucosal inflammatory cytokines. The increased frequency of CD4+ lymphocytes in the spleen and lamina propria with dextran sodium sulphate induction was decreased in miR-155(-/-) mice. Similarly, miR-155 deficiency abrogated the increased numbers of interferon-γ expressing CD4+ T cells typically observed in wild-type mice in this model. The frequency of systemic and mucosal T helper type 17-, CCR9-expressing CD4+ T cells was also reduced in miR-155(-/-) mice compared with control mice. These findings strongly support a role for miR-155 in facilitating pro-inflammatory cellular responses in this model of IBD. Loss of miR-155 also results in decreases in T helper type 1/type 17, CD11b+) and CD11c+ cells, which correlated with reduced clinical scores and severity of disease. miR-155 may serve as a potential therapeutic target for the treatment of IBD.

MicroRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis.

MicroRNAs (miRNAs) play important roles in the regulation of immune responses. There is evidence that miRNAs also participate in the pathogenesis of multiple sclerosis (MS), but how the miRNAs regulate the pathogenesis of MS is still under investigation. The identification of new members of the miRNA family associated with the pathogenesis of MS could facilitate early diagnosis and treatment. Here, we show that the level of miRNA let-7e is significantly upregulated in EAE, an animal model of MS using miRNA array and quantitative real-time PCR. The expression of let-7e was mainly in CD4(+) T cells and infiltrated mononuclear cells of CNS, and highly correlated with the development of EAE. We found that let-7e silencing in vivo inhibited encephalitogenic Th1 and Th17 cells and attenuated EAE, with reciprocal increase of Th2 cells; overexpression of let-7e enhanced Th1 and Th17 cells and aggravated EAE. We also identified IL-10 as one of the functional targets of let-7e. Together, we propose that let-7e is a new miRNA involved in the regulation of encephalitogenic T-cell differentiation and the pathogenesis of EAE.

Role of microRNAs in resveratrol-mediated mitigation of colitis-associated tumorigenesis in Apc(Min/+) mice.

The pleiotropic effects of resveratrol include anti-inflammatory, antioxidant, and anticancer activities, and thus unique possibilities exist to explore mechanistic pathways of chemoprevention. The aim of this study was to investigate the role of microRNA (miRNA) alterations induced by resveratrol in the context of chemopreventive mechanisms against dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis in the Apc(Min/+) mouse. To that end, Apc(Min/+) mice were exposed to 2% DSS to enhance intestinal inflammation and polyp development. Concurrently, mice received either vehicle or resveratrol treatment via oral gavage for 5 weeks. Interestingly, treatment of DSS-exposed mice with resveratrol resulted in decreased number and size of polyps, fewer histologic signs of cell damage, and decreased proliferating epithelial cells in intestinal mucosa compared with vehicle. Resveratrol treatment dramatically reversed the effects of DSS on the numbers of specific inflammatory CD4(+) T cells, CD8(+) T cells, B cells, natural killer T cells, and myeloid-derived suppressor cells in mesenteric lymph nodes. Resveratrol treatment also decreased interleukin-6 (IL-6) and tumor necrosis factor-α protein levels and reduced IL-6 and cyclooxygenase-2 mRNA expression. Microarray analysis revealed 104 miRNAs exhibiting >1.5-fold differences in expression in the intestinal tissue of resveratrol-treated mice. Among them, two miRNAs with anti-inflammatory properties, miRNA-101b and miRNA-455, were validated to be upregulated with resveratrol treatment by reverse-transcription polymerase chain reaction. Pathway analysis revealed that numerous differentially regulated miRNAs targeted mRNAs associated with inflammatory processes with known roles in intestinal tumorigenesis. These results suggest that resveratrol mediates anti-inflammatory properties and suppresses intestinal tumorigenesis through miRNA modulation.