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

Chemerin signalling in adipose tissue and intestinal homeostasis

Chemerin is an adipokine and potent chemoattractant for cells expressing chemokine-like receptor 1 (CMKLR1) which plays important roles in metabolism and immunity. Chemerin is secreted as inactive prochemerin and undergoes extracellular processing to generate a variety of isoforms that range in bioactivity. Clinical studies have demonstrated that chemerin levels are positively associated with obesity and inflammatory disorders. However, the role of chemerin signalling in adipose tissue, and how elevated chemerin levels affect inflammatory disease progression, are poorly understood. In this thesis, I describe the development of techniques to modulate levels of chemerin signalling. I then investigate the role of chemerin signalling in three physiological contexts: mature adipocytes, pathogenesis of colitis, and intestinal microbiome composition. Using a microarray-based approach, I identified matrix metalloproteinase (MMP)3 and several chemokines as novel targets of chemerin signalling in adipocytes. Decreased chemerin levels resulted in increased MMP activity and macrophage migration towards adipocyte-conditioned media, via an NFkB-dependent mechanism. This suggests that elevated chemerin levels with obesity influence adipose tissue remodelling. Importantly, these effects were mediated through a unique adipocyte-processed chemerin isoform. I next demonstrated that local expression, secretion, and processing of chemerin were positively associated with colonic inflammation in a colitis model. CMKLR1 knockout (KO) mice developed signs of clinical illness more slowly than wildtype, but ultimately developed similar levels of inflammation. Intraperitoneal injection of bioactive chemerin had no effect on colitis severity, suggesting that local chemerin levels have a greater impact on the pathogenesis of colitis. Finally, I investigated differences in intestinal microbiome composition between wildtype, chemerin KO, and CMKLR1 KO mice. Significant differences in Akkermansia and Prevotella abundance were observed in the absence of CMKLR1. These bacterial populations are known to correlate with adiposity and glucose homeostasis, indicating that chemerin signalling might influence metabolic processes through modulation of microbiome composition. Considered altogether, this work demonstrates that local chemerin signalling and processing plays autocrine/paracrine roles in adipose tissue and intestinal homeostasis. Additionally, chemerin represents a novel biomarker in colon inflammation. Modulation of local, context-specific chemerin signalling therefore represents a potential therapeutic target for the treatment of metabolic and inflammatory processes

Drugs or diet? - Developing novel therapeutic strategies targeting the free fatty acid family of GPCRs

Free fatty acids (FFAs) are metabolic intermediates that may be obtained through the diet, synthesized endogenously, or produced via fermentation of carbohydrates by gut microbiota. In addition to serving as an important source of energy, FFAs are known to produce a variety of both beneficial and detrimental effects on metabolic and inflammatory processes. While historically, FFAs were believed to produce these effects only through intracellular targets such as peroxisome proliferator-activated receptors, it has now become clear that FFAs are also agonists for several GPCRs, including a family of four receptors now termed FFA1-4. Increasing evidence suggests that FFA1-4 mediate many of the beneficial properties of FFAs and not surprisingly, this has generated significant interest in the potential of these receptors as therapeutic targets for the treatment of a variety of metabolic and inflammatory disorders. In addition to the traditional strategy of developing small-molecule therapeutics targeting these receptors, there has also been some consideration given to alternate therapeutic approaches, specifically by manipulating endogenous FFA concentrations through alteration of either dietary intake, or production by gut microbiota. In this review, the current state of knowledge for FFA1-4 will be discussed, together with their potential as therapeutic targets in the treatment of metabolic and inflammatory disorders. In particular, the evidence in support of small molecule versus dietary and microbiota-based therapeutic approaches will be considered to provide insight into the development of novel multifaceted strategies targeting the FFA receptors for the treatment of metabolic and inflammatory disorders

Regulation of BMP-dependent chondrogenesis in early limb mesenchyme by TGFβ signals

In the developing axial skeleton, sequential sonic hedgehog (SHH) and bone morphogenetic protein (BMP) signals are required for specification of a chondrogenic fate in presomitic tissue. A similar paradigm is thought to operate in the limb, but the signals involved are unclear. To investigate the nature of these signals, we examined BMP action in mesenchymal populations derived from the early murine limb bud (approximately embryonic day 10.5). These populations exhibited a graded response to BMPs, in which early limb mesenchymal cells (from the distal hind limb) displayed an anti-chondrogenic response, whereas BMPs promoted chondrogenesis in more mature cell populations (from the proximal fore limb). Under these conditions, multiple Gata genes were induced by BMPs and the extent of induction correlated with BMP anti-chondrogenic activity. A screen of limb-bud-expressed ligands revealed that prior short-term exposure to transforming growth factor β1 (TGFβ1) ameliorated the anti-chondrogenic response to BMP. Furthermore, brief activation of the TGFβ pathway was found to be necessary for subsequent induction of chondrogenesis by BMPs. Our findings indicate that, similar to axial skeletogenesis, induction of chondrogenesis in the appendicular skeleton is a two-step process. However, the programs differ in the transient signals driving chondrogenic responsiveness to BMPs, with SHH operating in the former and TGFβ activation in the latter.</jats:p