Diagnostic and therapeutic potential of RNASET2 in Crohn's disease: Disease-risk polymorphism modulates allelic-imbalance in expression and circulating protein levels and recombinant-RNASET2 attenuates pro-inflammatory cytokine secretion.

Ribonuclease T2 gene (RNASET2) variants are associated in genome wide association studies (GWAS) with risk for several autoimmune diseases, including Crohn's disease (CD). In T cells, a functional and biological relationship exists between TNFSF15-mediated enhancement of IFN-γ production, mucosal inflammation and RNASET2. Disease risk variants are associated with decreased mRNA expression and clinical characteristics of severe CD; however, functional classifications of variants and underlying molecular mechanisms contributing to pathogenesis remain largely unknown. In this study we demonstrate that allelic imbalance of RNASET2 disease risk variant rs2149092 is associated with transcriptional and post-transcriptional mechanisms regulating transcription factor binding, promoter-transactivation and allele-specific expression. RNASET2 mRNA expression decreases in response to multiple modes of T cell activation and recovers following elimination of activator. In CD patients with severe disease necessitating surgical intervention, preoperative circulating RNASET2 protein levels were decreased compared to non-IBD subjects and rebounded post-operatively following removal of the inflamed region, with levels associated with allelic carriage. Furthermore, overexpression or treatment with recombinant RNASET2 significantly reduced IFN-γ secretion. These findings reveal that RNASET2 cis- and trans-acting variation contributed regulatory complexity and determined expression and provide a basis for linking genetic variation with CD pathobiology. These data may ultimately identify RNASET2 as an effective therapeutic target in a subset of CD patients with severe disease.

Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn's Disease.

BACKGROUND & AIMS: Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). METHODS: Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. RESULTS: We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1 × 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. CONCLUSIONS: We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

Targeting metabolic plasticity in breast cancer cells via mitochondrial complex I modulation.

Heterogeneity commonly observed in clinical tumors stems both from the genetic diversity as well as from the differential metabolic adaptation of multiple cancer types during their struggle to maintain uncontrolled proliferation and invasion in vivo. This study aims to identify a potential metabolic window of such adaptation in aggressive human breast cancer cell lines. With a multidisciplinary approach using high-resolution imaging, cell metabolism assays, proteomic profiling and animal models of human tumor xenografts and via clinically-relevant pharmacological approach for modulating mitochondrial complex I function in human breast cancer cell lines, we report a novel route to target metabolic plasticity in human breast cancer cells. By a systematic modulation of mitochondrial function and by mitigating metabolic switch phenotype in aggressive human breast cancer cells, we demonstrate that the resulting metabolic adaptation signatures can predictably decrease tumorigenic potential in vivo. Proteomic profiling of the metabolic adaptation in these cells further revealed novel protein-pathway interactograms highlighting the importance of antioxidant machinery in the observed metabolic adaptation. Improved metabolic adaptation potential in aggressive human breast cancer cells contribute to improving mitochondrial function and reducing metabolic switch phenotype-which may be vital for targeting primary tumor growth in vivo.

Functional signaling of membrane-bound TL1A induces IFN-gamma expression.

TL1A, a TNF member implicated in autoimmune diseases, is a transmembrane protein that is processed to release soluble TL1A (TL1A-S). TL1A-S induces a Th1 response, although the functional significance of membrane-bound TL1A (TL1A-M) remains unknown. We generated TL1A-M expression in HEK-293 cells capable of binding DR3-Fc. Co-incubating IL-12/IL-18-primed CD4(+) T cells with HEK-293 cells expressing TL1A-M induced 3-fold increase in IFN-gamma that was blocked by anti-TL1A Ab. These results demonstrate that TL1A-M can bind death domain receptor 3 (DR3) through cell-cell contact to induce downstream IFN-gamma secretion enhancement. Anti-TL1A antibodies designed to treat immune diseases should be verified to block both endogenous TL1A forms.

Spectral kinetics ratiometry: a simple approach for real-time monitoring of fluorophore distributions in living cells.

BACKGROUND: Spectral Imaging Microscopy is gaining attention in biological research. Most of the commercial systems in vogue employ linear spectral un-mixing algorithms and/or spectral profile matching algorithms to extract the component spectral information from the measured specimen spectra. The need to accurately deconvolve multiple spectra with minimal cross-contamination is always accompanied by an increase in system complexity and cost. METHODS: We describe here a variant of the spectral waveform cross-correlation analysis (SWCCA) method where the master reference spectral library is constructed by composite spectra with varying ratios of component spectra, unlike the conventional spectral library where pure spectra form the components. We demonstrate that this spectral kinetics ratiometric approach gives realistic estimates of fluorophore distribution in living cells with a better spectral correlation as compared with pure component spectral libraries. RESULTS: Biological applications demonstrated in this article include acceptor photobleaching FRET, caspase activity during cell death and mitochondrial membrane polarization kinetics during substrate metabolism. CONCLUSIONS: Beyond the representative applications presented in this article, we think the proposed approach can be valuable in dynamic studies of a variety of other cellular processes such as pH oscillations, photobleaching and quenching kinetics. Besides giving better spectral correlation and real-time monitoring of biophysical processes in living cells, this method can serve as an economical solution for high-throughput spectral classification requirements.

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.