Glucocorticoid-induced microRNA-511 protects against TNF by down-regulating TNFR1

TNF is a central actor during inflammation and a well-recognized drug target for inflammatory diseases. We found that the mouse strain SPRET/Ei, known for extreme and dominant resistance against TNF-induced shock, displays weak expression of TNF receptor 1 protein (TNFR1) but normal mRNA expression, a trait genetically linked to the major TNFR1 coding gene Tnfrsf1a and to a locus harbouring the predicted TNFR1-regulating miR-511. This miRNA is a genuine TNFR1 regulator in cells. In mice, overexpression of miR-511 down-regulates TNFR1 and protects against TNF, while anti-miR-511 up-regulates TNFR1 and sensitizes for TNF, breaking the resistance of SPRET/Ei. We found that miR-511 inhibits endotoxemia and experimental hepatitis and that this miR is strongly induced by glucocorticoids and is a true TNFR1 modulator and thus an anti-inflammatory miR. Since minimal reductions of TNFR1 have considerable effects on TNF sensitivity, we believe that at least part of the anti-inflammatory effects of glucocorti-coids are mediated by induction of this miR, resulting in reduced TNFR1 expression

LPS resistance of SPRET/Ei mice is mediated by Gilz, encoded by the Tsc22d3 gene on the X chromosome

Natural variation for LPS-induced lethal inflammation in mice is useful for identifying new genes that regulate sepsis, which could form the basis for novel therapies for systemic inflammation in humans. Here we report that LPS resistance of the inbred mouse strain SPRET/Ei, previously reported to depend on the glucocorticoid receptor (GR), maps to the distal region of the X-chromosome. The GR-inducible gene Tsc22d3, encoding the protein Gilz and located in the critical region on the X-chromosome, showed a higher expressed SPRET/Ei allele, regulated in cis. Higher Gilz levels were causally related to reduced inflammation, as shown with knockdown and overexpression studies in macrophages. Transient overexpression of Gilz by hydrodynamic plasmid injection confirmed that Gilz protects mice against endotoxemia Our data strongly suggest that Gilz is responsible for the LPS resistance of SPRET/Ei mice and that it could become a treatment option for sepsis

Increased glucocorticoid receptor expression and activity mediate the LPS resistance of SPRET/EI mice

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared to C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent antiinflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared to C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased and insulin secretion from pancreatic β-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique since they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism

Description and mapping of the resistance of DBA/2 mice to TNF-induced lethal shock

In our search for genes that inhibit the inflammatory effects of TNF without diminishing its antitumor capacities we found that, compared with C57BL/6 mice, DBA/2 mice exhibit a dominant resistance to TNF-induced lethality. Tumor-bearing (C57BL/6 X DBA/2)(BXD)F-1 mice completely survived an otherwise lethal TNF/IFN-gamma-antitumor therapy with complete regression of the tumor. This was not the case for C57BL/6 mice. Genetic linkage analysis revealed that TNF resistance is linked to a major locus on distal chromosome 6 and a minor locus on chromosome 17. Compared with littermate controls, chromosome substitution mice carrying a DBA/2 chromosome 6 in a C57BL/6 background were significantly protected against TNF and TNF/IFN-gamma, albeit less so than DBA/2 mice. Definition of a critical region of 13 Mb on chromosome 6 was the highest mapping resolution obtained. Further analysis of candidate genes may provide a powerful tool to control TNF-induced pathologies in humans

Breaking the species barrier: derivation of germline-competent embryonic stem cells from Mus spretus x C57BL/6 hybrids

Embryonic stem (ES) cells, which can differentiate into almost all types of cells, have been derived from the house mouse Mus musculus, rat, rabbit, humans, and other species. Transmission of the genotype to the offspring of chimeras has been achieved only with M. musculus ES cells. limiting targeted mutagenesis using ES cells to this species. Mus spretus, which exhibits many genetic polymorphisms with M. musculus, displays dominant resistance to cancer and inflammation, making derived inbred strains very useful in positional cloning and interspecies mapping. We show here for the first time the derivation of ES cells from hybrid blastocysts, obtained by the mating of two different species, namely Mus muscalus and Mus spretus, and their use for the generation of chimeric mice that transmit the Mus spretus genotype and phenotype to the offspring. These hybrid ES cells allow the genetic manipulation of Mus spretus, as an alternative to Mus musculus

Determining differentially expressed miRNAs and validating miRNA-target relationships using the SPRET/Ei mouse strain

Micro RNAs (miRs) are involved in many biological processes. The challenge of identifying genes influenced by miRs is evidenced by the relatively few validated miR-target interactions. In this work, we used the Mus spretus SPRET/Ei strain as an in vivo system to identify new miR-target relations. Mus spretus diverged from Mus musculus over one million years ago, making it genetically and phenotypically divergent. SPRET/Ei mice are resistant to inflammation and several cancers, making them attractive for different research fields. Their phenotype is unique and is considerably different from that of almost all other laboratory mouse strains. We exploited the characteristics of SPRET/Ei mice as a tool to identify miR-target relationships. Hepatic genes and miRs differentially expressed between C57BL/6 and SPRET/Ei mice at basal levels were identified with an Affymetrix microarray and a multiplex qPCR, respectively. A total of 955 genes and 38 miRs were identified as differentially expressed. Increased miR expression might result in downregulation of its target mRNA and vice versa. Subsequently, we used our miR and mRNA data to identify possible in vivo miR-target interactions. Ingenuity pathway analysis (IPA) analysis revealed 380 possible miR-target interactions. Five miRs were selected for experimental validation by in vivo overexpression of the miRs. This resulted in the confirmation of six previously unknown miR-target interactions: miR-146a, Zdhhc2; miR-150, Elovl3, Kcnk5, and Nrd1d2; miR-155, Camta1; and miR-592, Steap2. In conclusion, we show that SPRET/Ei mice can be used as a platform for miR-target identification in vivo, and we used this platform to identify and experimentally confirm miR-target interactions

Response of TNF-hyporesponsive SPRET/Ei mice in models of inflammatory disorders

Most inflammatory disorders are becoming more prevalent, especially in Western countries. The proinflammatory cytokine tumor necrosis factor-alpha (TNF) plays a prominent role in many of these inflammatory disorders. We have previously shown that SPRET/Ei mice exhibit an extreme and dominant resistance to high doses of TNF. In this report, we investigate the response of heterozygous (C57BL/6xSPRET/Ei)F1 mice in different models of inflammatory diseases. Compared with C57BL/6 mice, (B x S)F1 mice are protected against TNF-induced arthritis and are partially protected against allergic asthma in an ovalbumin-induced model. However, these mice display complete susceptibility to TNF-induced inflammatory bowel disease. These results indicate that the SPRET/Ei genome harbors potent dominant antiinflammatory genes that might be relevant for the treatment of certain chronic inflammatory diseases. It is very well possible that different genes are implicated in the different models