The impact of chemerin or chemokine-like receptor 1 loss on the mouse gut microbiome

Chemerin is an adipocyte derived signalling molecule (adipokine) that serves as a ligand activator of Chemokine-like receptor 1(CMKLR1). Chemerin/CMKLR1 signalling is well established to regulate fundamental processes in metabolism and inflammation. The composition and function of gut microbiota has also been shown to impact the development of metabolic and inflammatory diseases such as obesity, diabetes and inflammatory bowel disease. In this study, we assessed the microbiome composition of fecal samples isolated from wildtype, chemerin, or CMKLR1 knockout mice using Illumina-based sequencing. Moreover, the knockout mice and respective wildtype mice used in this study were housed at different universities allowing us to compare facility-dependent effects on microbiome composition. While there was no difference in alpha diversity within samples when compared by either facility or genotype, we observed a dramatic difference in the presence and abundance of numerous taxa between facilities. There were minor differences in bacterial abundance between wildtype and chemerin knockout mice, but significantly more differences in taxa abundance between wildtype and CMKLR1 knockout mice. Specifically, CMKLR1 knockout mice exhibited decreased abundance of Akkermansia and Prevotella, which correlated with body weight in CMKLR1 knockout, but not wildtype mice. This is the first study to investigate a linkage between chemerin/CMKLR1 signaling and microbiome composition. The results of our study suggest that chemerin/CMKLR1 signaling influences metabolic processes through effects on the gut microbiome. Furthermore, the dramatic difference in microbiome composition between facilities might contribute to discrepancies in the metabolic phenotype of CMKLR1 knockout mice reported by independent groups. Considered altogether, these findings establish a foundation for future studies to investigate the relationship between chemerin signaling and the gut microbiome on the development and progression of metabolic and inflammatory disease

Retinoid Regulation of Antiviral Innate Immunity in Hepatocytes

Persistent infection of hepatitis C virus (HCV) is one of the leading causes of end-stage liver disease (ESLD), such as decompensated cirrhosis and liver cancer. Of particular note, nearly half of HCV-infected people in the United States are reported to be heavy drinkers. This particular group of patients is known to rapidly progress to the ESLD. Although accelerated disease progression among alcohol abusers infected with HCV is clinically well recognized, the molecular pathophysiology behind this manifestation has not been well elucidated. Hepatocytes metabolize ethanol (EtOH) primarily through two steps of oxidative catabolism in which alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) play central roles. The ADHALDH pathway also governs the metabolism of retinol (vitamin A) to its transcriptionally active metabolite, retinoic acid (RA). In this study, we defined that the ADH-ALDH pathway serves as a potent antiviral host factor in hepatocytes, which regulates the expression of interferon (IFN)-stimulated genes (ISGs) by biogenesis of RA. ISGs constitute over 300 antiviral effectors, which cooperatively govern intracellular antiviral innate immunity. Our study revealed that intracellular RA levels greatly influence ISG expression under basal conditions. Moreover, RA augments ISG induction in response to viral infection or exposure to IFN in a gene-specific manner. Lastly, our results demonstrated that EtOH attenuates the antiviral function of the ADH-ALDH pathway, which suggests the possibility that EtOH-retinol metabolic competition is one of the molecular mechanisms for the synergism between HCV and alcohol abuse in liver disease progression. Conclusions: RA plays a critical role in the regulation of intracellular antiviral innate immunity in hepatocytes

Molecular and cellular characterization of the CYP26b1-null limb phenotype

Cyp26b1, a retinoic acid (RA)-metabolizing enzyme, is expressed in the developing limb bud and Cyp26b1-/- mice present with severe early limb defects characterized by truncated skeletal elements and oligodactyly. These malformations have previously been attributed to a patterning defect; however, recent reports suggest that RA is dispensable for limb patterning. In this study, we examined the role of endogenous retinoid signalling in skeletogenesis using Cyp26b1-/- mice and transgenic mice in which Cyp26b1 is conditionally deleted under control of the Prrx1 promoter beginning at ~E9.5 (Prrx1Cre⁺/Cyp26b1fl/fl). We found that the limb phenotype in Prrx1Cre⁺/Cyp26b1fl/fl mice was less severe than Cyp26b1-/- animals and that a difference in retinoid signalling contributed to the difference in phenotypes. We systematically examined the role of RA signalling in chondrogenesis and found that Cyp26b1-/- cells are maintained at a pre-chondrogenic stage, exhibit reduced chondroblast differentiation, and exhibit a modest impact on chondrocyte hypertrophy. Furthermore, Cyp26b1-/- mesenchyme exhibited an increase in the expression of Scleraxis and other tendon markers, indicating that increased retinoid signalling in the limb maintains the ability of precursor cells to commit to other mesenchymal lineages. We conclude that RA signalling negatively impacts chondrogenesis before the onset of Ihh signalling, and has a positive impact on chondrocyte hypertrophy. This suggests that the limb phenotype in Cyp26b1-/- animals results from defects in the execution of a patterning program, and not so much in the patterning program itself.Medicine, Faculty ofGraduat

Analysis of chondrogenesis using micromass cultures of limb mesenchyme

High-density micromass cultures of embryonic mesenchymal cells have proved to be an invaluable model for studying the entire chondrogenic program, from precartilaginous condensations through to chondrocyte hypertrophy. This culture model also provides a powerful system in which to explore the function of various factors in the commitment and differentiation of mesenchymal cells to the chondrogenic lineage. In this regard, micromass cultures provide a consistent and robust model for investigating the effects of genetic manipulations on skeletal phenotypes and for delineating their molecular basis. In this methods chapter, the derivation and use of micromass cultures from murine limb buds are described, but these techniques are also applicable to other organisms and mesenchymal cell sources. © 2014 Springer Science+Business Media, LLC