Single cell analysis shows decreasing FoxP3 and TGFβ1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes

Natural CD4+CD25+ regulatory T (CD4+CD25+ T reg) cells play a key role in the immunoregulation of autoimmunity. However, little is known about the interactions between CD4+CD25+ T reg cells and autoreactive T cells. This is due, in part, to the difficulty of using cell surface markers to identify CD4+CD25+ T reg cells accurately. Using a novel real-time PCR assay, mRNA copy number of FoxP3, TGFβ1, and interleukin (IL)-10 was measured in single cells to characterize and quantify CD4+CD25+ T reg cells in the nonobese diabetic (NOD) mouse, a murine model for type 1 diabetes (T1D). The suppressor function of CD4+CD25+CD62Lhi T cells, mediated by TGFβ, declined in an age-dependent manner. This loss of function coincided with a temporal decrease in the percentage of FoxP3 and TGFβ1 coexpressing T cells within pancreatic lymph node and islet infiltrating CD4+CD25+CD62Lhi T cells, and was detected in female NOD mice but not in NOD male mice, or NOR or C57BL/6 female mice. These results demonstrate that the majority of FoxP3-positive CD4+CD25+ T reg cells in NOD mice express TGFβ1 but not IL-10, and that a defect in the maintenance and/or expansion of this pool of immunoregulatory effectors is associated with the progression of T1D

Promoter de-methylation of cyclin D2 by sulforaphane in prostate cancer cells

Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC) enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs), especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis

Effects of low zinc status and arsenic exposure in mice

During fall term of 2015 and winter and spring term of 2016 I assisted in research within Dr. Emily Ho’s lab. The project that I assisted in looked at the effects of low zinc status and arsenic exposure in mice. Zinc is an essential micronutrient involved in numerous cellular processes including its role as an antioxidant. Arsenic is a naturally occurring metal found within the earth’s crust. Humans can come into contact with arsenic through contaminated food and water sources. Arsenic exposure is associated with an increase in reactive oxygen species. The goal of our study was to determine if zinc deficiency or arsenic exposure caused an increase in oxidative damage. For this study we used 5 groups 8 mice. The mice were randomly assigned to either a zinc adequate group, which were fed a diet containing zinc equivalent to RDA recommendations, or a zinc deficient group, which was fed a diet containing very low zinc levels. Mice were also randomly assigned to 3 groups which contained 0 ppb, 50 ppb and 500 ppb arsenic in their drinking water. The mice received the assigned treatments for 4 weeks. After the conclusion of the treatment, mineral levels of calcium, copper, iron, magnesium, selenium and zinc were determined using ICP-OES (Inductively coupled plasma optical emission spectrometry). Liver damage was evaluated by examining plasma ALT (alanine aminotransferase) levels which is a biomarker for liver damage. Oxidative DNA damage was evaluated in liver genomic DNA using 8-OHdG DNA damage ELISA. We determined that arsenic exposure reduced magnesium and selenium content within plasma. Both zinc deficiency and arsenic exposure increased levels of 8-OHdG, and zinc deficiency increased levels of ALT

Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation

Aging is a complex process associated with physiological changes in numerous organ systems. In particular, aging of the immune system is characterized by progressive dysregulation of immune responses, resulting in increased susceptibility to infectious diseases, impaired vaccination efficacy, as well as systemic low grade chronic inflammation. Increasing evidence suggest that intracellular zinc homeostasis, regulated by zinc transporter expression, is critically involved in the signaling and activation of immune cells. We hypothesize that epigenetic alterations and nutritional deficits associated with aging may lead to zinc transporter dysregulation, resulting in decreases in cellular zinc levels and enhanced inflammation with age. The goal of this study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells. The effects of zinc deficiency and age on the induction of inflammatory responses were determined using an in vitro cell culture system as well as an aged mouse model. We showed that zinc deficiency, particularly the reduction in intracellular zinc in immune cells, was associated with increased inflammation with age. Furthermore, reduced Zip 6 expression enhanced proinflammatory response, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Furthermore, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our data suggested that age-related epigenetic dysregulation in zinc transporter expression may influence cellular zinc levels and contribute to increased susceptibility to inflammation with age.This is the authors' peer-reviewed accepted manuscript. Version of record is copyrighted by Elsevier and can be found at: http://www.journals.elsevier.com/the-journal-of-nutritional-biochemistry

Effects of Promotional Materials on Attitudes and Fear towards Colorectal Cancer Screening among Chinese Older Adults: An Experimental Study

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Growth Hormone Regulates the Balance Between Bone Formation and Bone Marrow Adiposity

Cancellous bone decreases and bone marrow fat content increases with age. Osteoblasts and adipocytes are derived from a common precursor, and growth hormone (GH), a key hormone in integration of energy metabolism, regulates the differentiation and function of both cell lineages. Since an age-related decline in GH is associated with bone loss, we investigated the relationship between GH and bone marrow adiposity in hypophysectomized (HYPOX) rats and in mice with defects in GH signaling. HYPOX dramatically reduced body weight gain, bone growth and mineralizing perimeter, serum insulin-like growth factor 1 (IGF-1) levels, and mRNA levels for IGF-1 in liver and bone. Despite reduced body mass and adipocyte precursor pool size, HYPOX resulted in a dramatic increase in bone lipid levels, as reflected by increased bone marrow adiposity and bone triglyceride and cholesterol content. GH replacement normalized bone marrow adiposity and precursor pool size, as well as mineralizing perimeter in HYPOX rats. In contrast, 17β -estradiol, IGF-1, thyroxine, and cortisone were ineffective. Parathyroid hormone (PTH) reversed the inhibitory effects of HYPOX on mineralizing perimeter but had no effect on adiposity. Finally, bone marrow adiposity was increased in mice deficient in GH and IGF-1 but not in mice deficient in serum IGF-1. Taken together, our findings indicate that the reciprocal changes in bone and fat mass in GH signaling-deficient rodents are not directly coupled with one another. Rather, GH enhances adipocyte as well as osteoblast precursor pool size. However, GH increases osteoblast differentiation while suppressing bone marrow lipid accumulation. © 2010 American Society for Bone and Mineral Researc

Docosahexaenoic acid attenuates Western diet-induced hepatic fibrosis in Ldlr(-/-) mice by targeting the TGFβ-Smad3 pathway

DHA (22:6,ω3), but not EPA (20:5,ω3), attenuates Western diet (WD)-induced hepatic fibrosis in a Ldlr[superscript −/−] mouse model of nonalcoholic steatohepatitis. We examined the molecular basis for the differential effect of dietary EPA and DHA on WD-induced hepatic fibrosis. DHA was more effective than EPA at preventing WD-induced effects on hepatic transcripts linked to fibrosis, including collagen 1A1 (Col1A1), transforming growth factor-β (TGFβ) signaling and proteins involved in remodeling the extracellular matrix, including metalloproteases, tissue inhibitors of metalloproteases, and lysyl oxidase subtypes. Examination of the TGFβ pathway showed that mice fed the WD supplemented with either olive oil or EPA had a significant (≥2.5-fold) increase in hepatic nuclear abundance of phospho-mothers against decapentaplegic homolog (Smad)3 when compared with mice fed the reference diet (RD); Smad3 is a key regulator of Col1A1 expression in stellate cells. In contrast, mice fed the WD supplemented with DHA had no increase in phospho-Smad3 when compared with mice fed the RD. Changes in hepatic phospho-Smad3 nuclear content correlated with proCol1A1 mRNA and protein abundance. Pretreatment of human LX2 stellate cells with DHA, but not other unsaturated fatty acids, blocked TGFβ1-mediated induction of Col1A1. In conclusion, DHA attenuates WD-induced fibrosis by targeting the TGFβ-Smad3-Col1A1 pathway in stellate cells

Aging and serum MCP-1 are associated with gut microbiome composition in a murine model

Introduction. Age is the primary risk factor for major human chronic diseases, including cardiovascular disorders, cancer, type 2 diabetes, and neurodegenerative diseases. Chronic, low-grade, systemic inflammation is associated with aging and the progression of immunosenescence. Immunosenescence may play an important role in the development of age-related chronic disease and the widely observed phenomenon of increased production of inflammatory mediators that accompany this process, referred to as “inflammaging.” While it has been demonstrated that the gut microbiome and immune system interact, the relationship between the gut microbiome and age remains to be clearly defined, particularly in the context of inflammation. The aim of our study was to clarify the associations between age, the gut microbiome, and pro-inflammatory marker serum MCP-1 in a C57BL/6 murine model. Results. We used 16S rRNA gene sequencing to profile the composition of fecal microbiota associated with young and aged mice. Our analysis identified an association between microbiome structure and mouse age and revealed specific groups of taxa whose abundances stratify young and aged mice. This includes the Ruminococcaceae, Clostridiaceae, and Enterobacteriaceae. We also profiled pro-inflammatory serum MCP-1 levels of each mouse and found that aged mice exhibited elevated serum MCP-1, a phenotype consistent with inflammaging. Robust correlation tests identified several taxa whose abundance in the microbiome associates with serum MCP-1 status, indicating that they may interact with the mouse immune system. We find that taxonomically similar organisms can exhibit differing, even opposite, patterns of association with the host immune system. We also find that many of the OTUs that associate with serum MCP-1 stratify individuals by age. Discussion. Our results demonstrate that gut microbiome composition is associated with age and the pro-inflammatory marker, serum MCP-1. The correlation between age, relative abundance of specific taxa in the gut microbiome, and serum MCP-1 status in mice indicates that the gut microbiome may play a modulating role in age-related inflammatory processes. These findings warrant further investigation of taxa associated with the inflammaging phenotype and the role of gut microbiome in the health status and immune function of aged individuals.Keywords: Immunity, Immunosenescence, Mice, Microbiome, Inflammaging, Aging, InflammationThe following information was supplied regarding data availability: BioProject ID PRJNA311095, accessions SRX1581953, SRX1581958-RX1581960, and SRX1571787-RX1571792 http://files.cgrb.oregonstate.edu/Sharpton_Lab/Papers/Conley_PeerJ_2015