SUMMARY Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNA interference and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced co-activator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51؊/؊ mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.

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We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14α-demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arrest, senescence-associated secretory phenotype and ultimately the oval cell response, while elevated CYP51 substrates promoted the integrated stress response. In spite of the oval cell-driven fibrosis being histologically similar in both sexes, data indicates a female-biased down-regulation of primary metabolism pathways and a stronger immune response in males. Liver injury was ameliorated by dietary fats predominantly in females, whereas dietary cholesterol rectified fibrosis in both sexes. Our data place defective cholesterol synthesis as a focus of sex-dependent liver pathologies.

Seventy-four nonrepetitive uropathogenic fluoroquinolone-resistant or -intermediate extended-spectrum-␤-lactamase-producing Klebsiella isolates from Slovenia were screened for the presence of plasmid-mediated quinolone resistance genes. None of the known qnr genes were detected. The aac(6)-Ib-cr allele was detected on plasmids from 25 transconjugants for which the ciprofloxacin MIC was higher than for the recipient Escherichia coli strain.Fluoroquinolones are broad-spectrum antimicrobial agents widely used in clinical medicine. Resistance to this class of antibacterials is usually caused by mutations in the chromosomal genes that code for DNA gyrase and/or DNA topoisomerase IV, the target enzymes, and/or mutations resulting in alterations in drug accumulation (14). Recently, three plasmidmediated quinolone resistance (PMQR) mechanisms have also been described.The first comprises qnr genes that encode target protection proteins of the pentapeptide repeat family (8, 16). Three qnr genes, qnrA, qnrB, and qnrS, and their allelic variants have been described so far. Although the protein products of qnrA1 through qnrA5, qnrB1, qnrB2, qnrS1, and qnrS2 all provide low-level quinolone resistance, considerable variability can be observed in their amino acid sequences (2, 5, 7, 11).The second mechanism involves the aac(6Ј)-Ib-cr gene, which encodes a new variant of the common aminoglycoside acetyltransferase. Two single-nucleotide substitutions at codons 102 and 179 in the wild-type allele aac(6Ј)-Ib enable the gene product to be capable of acetylating and thus reducing the activity of some fluoroquinolones, including norfloxacin and ciprofloxacin (12).The prevalence of this PMQR gene has been so far reported from China, the United States, and Nigeria (9, 13, 15). Despite these recent findings, the contributions of the qnr and aac(6Ј)-Ib-cr genes to the increasing quinolone resistance worldwide and the association between quinolone resistance and extended-spectrum-␤-lactamase (ESBL)-producing strains remain largely unknown.The third mechanism, involving the fluoroquinolone efflux pump protein QepA, has been described very recently (17). The qepA gene was identified on plasmid pHPA of Escherichia coli C316. It resides on a putative transposable element, flanked by two copies of IS26. The similarity of QepA to the MFS-type pumps of the 14 TMS family, a high GϩC content, and the codon usage pattern indicate a probable intergeneric transfer of a qepA-like gene from environmental microbes to E. coli (17).Our study was initiated by the observation that the intermediate resistance phenotype, as determined by the CLSI (Clinical and Laboratory Standards Institute) guidelines (3), to the two fluoroquinolones most frequently used in Slovenia, ciprofloxacin and norfloxacin, almost disappeared in a collection of 74 Klebsiella isolates producing ESBLs, as detected by doubledisc synergy tests and interpreted according to CLSI criteria, from the Institute of Public Health of the Republic of Slovenia (IVZ) after 2003, although it was frequ...