MicroRNA-31 Targets Thymic Stromal Lymphopoietin in Mucosal Infiltrated CD4+ T Cells: A Role in Achieving Mucosal Healing in Ulcerative Colitis?

Background: Ulcerative colitis (UC) is characterized by disruption of the mucosal intestinal barrier. MicroRNAs, single-stranded noncoding RNAs of approximately 22nt, are dysregulated in UC. MicroRNAs targeting thymic stromal lymphopoietin (TSLP), a cytokine involved in T-cell maturation and polarization, may be involved in regulating UC inflammation and mucosal healing. Methods: Biopsy samples from non-UC (n = 38), inactive UC (n = 18), and active UC (n = 23) patients were analyzed for mRNA (real-time quantitative polymerase chain reaction) or TSLP protein expression (enzyme-linked immunosorbent assay). Flow cytometry was used to isolate CD4+ T cells from biopsies. The functional mechanism was shown using luciferase assays and antago-miR transfections. The TSLP/miR-31 association was analyzed on 196 subjects from a previous clinical trial that tested the anti-IL-13 drug tralokinumab, whereas mucosal healing effects were studied on a subset of patients (n = 13) from this trial. Results: We found that TSLP is reduced at both mRNA and protein levels in inflamed UC patients when compared with healthy subjects, in both whole biopsies and biopsy-isolated CD4+ CD25+ T cells. The expression of miR-31, predicted to target TSLP, inversely co-related to the levels of TSLP mRNA in T cells. Blocking miR-31 in vitro in T cells increased both TSLP mRNA expression and protein secretion. Luciferase assays showed that miR-31 directly targeted TSLP mRNA, suggesting a direct mechanistic link. We also found that TSLP is increased in patients who achieve mucosal healing, comparing biopsies before and after treatment from the tralokinumab trial. Conclusions: Our data suggest a role for TSLP in promoting mucosal healing and regulating inflammation in UC, whereas miR-31 can directly block this effect.

MicroRNA-31 and MicroRNA-155 Are Overexpressed in Ulcerative Colitis and Regulate IL-13 Signaling by Targeting Interleukin 13 Receptor α-1.

Interleukin-13 (IL-13) is an important Type 2 T helper (Th2) cytokine, controlling biological functions in epithelium and has been linked to asthma, atopic dermatitis and ulcerative colitis (UC). Interleukin-13 signals through IL-13 receptor α-1 (IL13RA1 (gene) and IL13Rα1 (protein)), a receptor that can be regulated by microRNAs (miRs). MicroRNAs are small non-coding single-stranded RNAs with a role in several pathologies. However, their relevance in the pathophysiology of UC, a chronic inflammatory condition of the colonic mucosa, is poorly characterised. Here, we determined the expression of IL13Rα1 in UC, its potential regulation by miRs and the subsequent effect on IL-13 signalling. Inflamed mucosa of UC patients showed decreased mRNA and protein expression of IL13RA1 when compared to healthy controls. We show that miR-31 and miR-155 are upregulated in inflamed UC mucosa and that both directly target the 3' untranslated region of IL13RA1 mRNA. Transfection of miR-31 and miR-155 mimics reduced the expression of IL13RA1 mRNA and protein, and blocked IL-13-dependent phosphorylation of signal transducer and activator of transcription 6 (STAT6) in HT-29 cells, a gut epithelium cell line. Interleukin-13 activation of suppressor of cytokine signaling 1 (SOCS1) and eotaxin-3 (CCL26) expression was also diminished. MicroRNA-31/microRNA-155 mimics also downregulated IL13RA1 in ex vivo human inflamed UC biopsies. We propose that miR-31 and miR-155 have an important role in limiting IL-13 signalling in UC disease.

Copper transporters are responsible for copper isotopic fractionation in eukaryotic cells.

Copper isotopic composition is altered in cancerous compared to healthy tissues. However, the rationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cu uptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enriched in 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transporters is of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotope fractionation is observed and when Cu is provided solely in reduced form for this strain, copper is enriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionation is generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on ab initio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich amino acids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3 copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative to the surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could be due to lower reductase activity in the tumor.

Medical applications of Cu, Zn, and S isotope effects.

This review examines recent applications of stable copper, zinc and sulfur isotopes to medical cases and notably cancer. The distribution of the natural stable isotopes of a particular element among coexisting molecular species varies as a function of the bond strength, the ionic charge, and the coordination, and it also changes with kinetics. Ab initio calculations show that compounds in which a metal binds to oxygen- (sulfate, phosphate, lactate) and nitrogen-bearing moieties (histidine) favor heavy isotopes, whereas bonds with sulfur (cysteine, methionine) favor light isotopes. Oxidized cations (e.g., Cu(ii)) and low coordination numbers are expected to favor heavy isotopes relative to their reduced counterparts (Cu(i)) and high coordination numbers. Here we discuss the first observations of Cu, Zn, and S isotopic variations, three elements closely related along multiple biological pathways, with emphasis on serum samples of healthy volunteers and of cancer patients. It was found that heavy isotopes of Zn and to an even greater extent Cu are enriched in erythrocytes relative to serum, while the difference is small for sulfur. Isotopic variations related to age and sex are relatively small. The 65Cu/63Cu ratio in the serum of patients with colon, breast, and liver cancer is conspicuously low relative to healthy subjects. The characteristic time over which Cu isotopes may change with disease progression (a few weeks) is consistent with both the turnover time of the element and albumin half-life. A parallel effect on sulfur isotopes is detected in a few un-medicated patients. Copper in liver tumor tissue is isotopically heavy. In contrast, Zn in breast cancer tumors is isotopically lighter than in healthy breast tissue. 66Zn/64Zn is very similar in the serum of cancer patients and in controls. Possible reasons for Cu isotope variations may be related to the cytosolic storage of Cu lactate (Warburg effect), release of intracellular copper from cysteine clusters (metallothionein), or the hepatocellular and biosynthetic dysfunction of the liver. We suggest that Cu isotope metallomics will help evaluate the homeostasis of this element during patient treatment, notably by chelates and blockers of Cu trafficking, and understand the many biochemical pathways in which this element is essential.

Hypoxia induces copper stable isotope fractionation in hepatocellular carcinoma, in a HIF-independent manner.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer, with increasing incidence worldwide. The unrestrained proliferation of tumour cells leads to tumour hypoxia which in turn promotes cancer aggressiveness. While changes in the concentration of copper (Cu) have long been observed upon cancerization, we have recently reported that the isotopic composition of copper is also altered in several types of cancer. In particular, we showed that in hepatocellular carcinoma, tumour tissue contains heavier copper compared to the surrounding parenchyma. However, the reasons behind such isotopic signature remained elusive. Here we show that hypoxia causes heavy copper enrichment in several human cell lines. We also demonstrate that this effect of hypoxia is pH, HIF-1 and -2 independent. Our data identify a previously unrecognized cellular process associated with hypoxia, and suggests that in vivo tumour hypoxia determines copper isotope fractionation in HCC and other solid cancers.

Copper isotope effect in serum of cancer patients. A pilot study.

The isotope effect describes mass-dependent variations of natural isotope abundances for a particular element. In this pilot study, we measured the (65)Cu/(63)Cu ratios in the serums of 20 breast and 8 colorectal cancer patients, which correspond to, respectively, 90 and 49 samples taken at different times with molecular biomarker documentation. Copper isotope compositions were determined by multiple-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). When compared with the literature data from a control group of 50 healthy blood donors, abundances of Cu isotopes predict mortality in the colorectal cancer group with a probability p = 0.018. For the breast cancer patients and the group of control women the probability goes down to p = 0.0006 and the AUC under the ROC curve is 0.75. Most patients considered in this preliminary study and with serum δ(65)Cu lower than the threshold value of -0.35‰ (per mil) did not survive. As a marker, a drop in δ(65)Cu precedes molecular biomarkers by several months. The observed decrease of δ(65)Cu in the serum of cancer patients is assigned to the extensive oxidative chelation of copper by cytosolic lactate. The potential of Cu isotope variability as a new diagnostic tool for breast and colorectal cancer seems strong. Shifts in Cu isotope compositions fingerprint cytosolic Cu chelation by lactate mono- and bidentates. This simple scheme provides a straightforward explanation for isotopically light Cu in the serum and isotopically heavy Cu in cancer cells: Cu(+) escaping chelation by lactate and excreted into the blood stream is isotopically light. Low δ(65)Cu values in serum therefore reveal the strength of lactate production by the Warburg effect.

A microRNA network dysregulated in asthma controls IL-6 production in bronchial epithelial cells.

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-ß, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-ß, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-ß and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.