Single-cell, single-nucleus, and spatial transcriptomics characterization of the immunological landscape in the healthy and PSC human liver.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which there is an unmet need to understand the cellular composition of the affected liver and how it underlies disease pathogenesis. We aimed to generate a comprehensive atlas of the PSC liver using multi-omic modalities and protein-based functional validation. METHODS: We employed single-cell and single-nucleus RNA sequencing (47,156 cells and 23,000 nuclei) and spatial transcriptomics (one sample by 10x Visium and five samples with Nanostring GeoMx DSP) to profile the cellular ecosystem in 10 PSC livers. Transcriptomic profiles were compared to 24 neurologically deceased donor livers (107,542 cells) and spatial transcriptomics controls, as well as 18,240 cells and 20,202 nuclei from three PBC livers. Flow cytometry was performed to validate PSC-specific differences in immune cell phenotype and function. RESULTS: PSC explants with parenchymal cirrhosis and prominent periductal fibrosis contained a population of cholangiocyte-like hepatocytes that were surrounded by diverse immune cell populations. PSC-associated biliary, mesenchymal, and endothelial populations expressed chemokine and cytokine transcripts involved in immune cell recruitment. Additionally, expanded CD4+ T cells and recruited myeloid populations in the PSC liver expressed the corresponding receptors to these chemokines and cytokines, suggesting potential recruitment. Tissue-resident macrophages, by contrast, were reduced in number and exhibited a dysfunctional and downregulated inflammatory response to lipopolysaccharide and interferon-γ stimulation. CONCLUSIONS: We present a comprehensive atlas of the PSC liver and demonstrate an exhaustion-like phenotype of myeloid cells and markers of chronic cytokine expression in late-stage PSC lesions. This atlas expands our understanding of the cellular complexity of PSC and has potential to guide the development of novel treatments. IMPACT AND IMPLICATIONS: Primary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic inflammation and irreparable damage to the bile ducts, which eventually results in liver failure. Due to a limited understanding of the underlying pathogenesis of disease, treatment options are limited. To address this, we sequenced healthy and diseased livers to compare the activity, interactions, and localization of immune and non-immune cells. This revealed that hepatocytes lining PSC scar regions co-express cholangiocyte markers, whereas immune cells infiltrate the scar lesions. Of these cells, macrophages, which typically contribute to tissue repair, were enriched in immunoregulatory genes and demonstrated a lack of responsiveness to stimulation. These cells may be involved in maintaining hepatic inflammation and could be a target for novel therapies.

A rat liver cell atlas reveals intrahepatic myeloid heterogeneity.

The large size and vascular accessibility of the laboratory rat (Rattus norvegicus) make it an ideal hepatic animal model for diseases that require surgical manipulation. Often, the disease susceptibility and outcomes of inflammatory pathologies vary significantly between strains. This study uses single-cell transcriptomics to better understand the complex cellular network of the rat liver, as well as to unravel the cellular and molecular sources of inter-strain hepatic variation. We generated single-cell and single-nucleus transcriptomic maps of the livers of healthy Dark Agouti and Lewis rat strains and developed a factor analysis-based bioinformatics analysis pipeline to study data covariates, such as strain and batch. Using this approach, we discovered transcriptomic variation within the hepatocyte and myeloid populations that underlie distinct cell states between rat strains. This finding will help provide a reference for future investigations on strain-dependent outcomes of surgical experiment models.

Impact of routine reporting of estimated glomerular filtration rate using the European Kidney Function Consortium and Chronic Kidney Disease Epidemiology Collaboration equations in a Western Australian community population.

AIM: In 2020, the European Kidney Function Consortium (EKFC) published a new creatinine-based equation to estimate glomerular filtration rate (eGFR) to overcome known limitations in existing equations. The aim of this study is to model the potential impact on service referral and health expenditure of routine reporting of eGFR using the EKFC equation as compared to the CKD-EPI equation in a Western Australian population. METHODS: eGFR was calculated for 760 614 patients with 2 368 234 creatinine results using the CKD-EPI and EKFC formulas. Patients were grouped into a CKD cohort if they had at least two eGFR results of <60 ml/min/1.73 m2 from tests at least 90 days apart. The impact of each equation on the reclassification of CKD stages, CKD cohort classification, the rate of change in eGFR and direct health costs were assessed. RESULTS: About 90.66% of patients had a lower eGFR when calculated using the EKFC equation. About 12.6% of individuals were classified into a different CKD stage using the EKFC equation with 97.43% of these patients classified into a higher (more advanced) stage. There was a 25.9% increase in the number of patients identified as having CKD when calculated using the EKFC equation. Direct health costs also increased with the use of EKFC reporting. CONCLUSION: Use of the EKFC equation will increase population prevalence of CKD and will result in a shift to higher stages of CKD. This has implications for monitoring and referral of patients within specialist services and has the potential to increase the need for multidisciplinary care.

Unlocking Potential within Health Systems Using Privacy-Preserving Record Linkage: Exploring Chronic Kidney Disease Outcomes through Linked Data Modelling.

BACKGROUND: Chronic kidney disease (CKD) is a major global health problem that affects approximately one in 10 adults. Up to 90% of individuals with CKD go undetected until its progression to advanced stages, invariably leading to death in the absence of treatment. The project aims to fill information gaps around the burden of CKD in the Western Australian (WA) population, including incidence, prevalence, rate of progression, and economic cost to the health system. METHODS: Given the sensitivity of the information involved, the project employed a privacy preserving record linkage methodology to link data from four major pathology providers in WA to hospital records, to establish a CKD registry with continuous medical record for individuals with biochemical specification for CKD. This method uses encrypted personal identifying information in a probability-based linkage framework (Bloom filters) to help mitigate risk while maximizing linkage quality. RESULTS: The project developed interoperable technology to create a transparent CKD data catalogue which is linkable to other datasets. This technology has been designed to support the aspirations of the research program to provide linked de-identified pathology, morbidity, and mortality data that can be used to derive insights to enable better CKD patient outcomes. The cohort includes over 1 million individuals with creatinine results over the period 2002 to 2021. CONCLUSION: Using linked data from across the care continuum, researchers are able to evaluate the effectiveness of service delivery and provide evidence for policy and program development. The CKD registry will enable an innovative review of the epidemiology of CKD in WA. Linking pathology records can identify cases of CKD that are missed in the early stages due to disaggregation of results, enabling identification of at-risk populations that represent targets for early intervention and management.

Prediction models used in the progression of chronic kidney disease: A scoping review.

OBJECTIVE: To provide a review of prediction models that have been used to measure clinical or pathological progression of chronic kidney disease (CKD). DESIGN: Scoping review. DATA SOURCES: Medline, EMBASE, CINAHL and Scopus from the year 2011 to 17th February 2022. STUDY SELECTION: All English written studies that are published in peer-reviewed journals in any country, that developed at least a statistical or computational model that predicted the risk of CKD progression. DATA EXTRACTION: Eligible studies for full text review were assessed on the methods that were used to predict the progression of CKD. The type of information extracted included: the author(s), title of article, year of publication, study dates, study location, number of participants, study design, predicted outcomes, type of prediction model, prediction variables used, validation assessment, limitations and implications. RESULTS: From 516 studies, 33 were included for full-text review. A qualitative analysis of the articles was compared following the extracted information. The study populations across the studies were heterogenous and data acquired by the studies were sourced from different levels and locations of healthcare systems. 31 studies implemented supervised models, and 2 studies included unsupervised models. Regardless of the model used, the predicted outcome included measurement of risk of progression towards end-stage kidney disease (ESKD) of related definitions, over given time intervals. However, there is a lack of reporting consistency on details of the development of their prediction models. CONCLUSIONS: Researchers are working towards producing an effective model to provide key insights into the progression of CKD. This review found that cox regression modelling was predominantly used among the small number of studies in the review. This made it difficult to perform a comparison between ML algorithms, more so when different validation methods were used in different cohort types. There needs to be increased investment in a more consistent and reproducible approach for future studies looking to develop risk prediction models for CKD progression.

Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity.

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single-cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation-related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at a single-cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte, and mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

Pancreas-specific SNAP23 depletion prevents pancreatitis by attenuating pathological basolateral exocytosis and formation of trypsin-activating autolysosomes.

Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKß-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKß inhibitors, and confirmed by the inability of IKBKB/IKKß phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKß's involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.Abbreviations: AL: autolysosome; AP: acute pancreatitis; AV: autophagic vacuole; CCK: cholecystokinin; IKBKB/IKKß: inhibitor of nuclear factor kappa B kinase subunit beta; SNAP23: synaptosome associated protein 23; SNARE: soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor; STX: syntaxin; TAP: trypsinogen activation peptide; VAMP: vesicle associated membrane protein; ZG: zymogen granule.

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

Nanoparticle Uptake in a Spontaneous and Immunocompetent Woodchuck Liver Cancer Model.

There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based in vivo experiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14+ cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.

Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.

The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment.

Incidence and predictors of HIV, chlamydia and gonorrhoea among men who have sex with men attending a peer-based clinic.

Introduction Despite a range of interventions, annual numbers of new diagnoses of HIV infection among men who have sex with men (MSM) in Australia have not declined in recent years. Peer-based sexual health clinics targeting MSM, such as the M Clinic in Perth (WA, Australia), have been put in place to provide safe sex counselling and to increase testing rates among MSM and who are at high risk of HIV infection. The aim of this study was to assess the incidence of HIV, chlamydia and gonorrhoea among men attending the M Clinic. METHODS: This was a historical cohort study of repeated M Clinic clients from January 2011 to June 2015 inclusive. Testing and risk factor data from M Clinic client software were used to estimate the incidence of HIV, chlamydia and gonorrhoea and associated factors. RESULTS: The incidence of HIV, chlamydia and gonorrhoea was 1.87, 13.58 and 6.48 per 100 person-years respectively. Older men had a higher incidence of HIV infection but a lower incidence of chlamydia and gonorrhoea than younger men. CONCLUSIONS: The HIV incidence was higher than found in similar studies in other Australian sexual health clinics, but the incidence of chlamydia and gonorrhoea was similar. The high HIV incidence among clients of the M Clinic points to the importance of making pre-exposure HIV prophylaxis available to clients of the M Clinic and similar services.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis from pooled rectal, pharyngeal and urine specimens in men who have sex with men.

OBJECTIVES: Screening of men who have sex with men (MSM) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) requires sampling from anorectal and pharyngeal sites in addition to urogenital sampling. Due to the cost of testing multiple anatomical sites individually testing of pooled specimens has potential merit. The Cepheid GeneXpert CT/NG assay (GeneXpert), which also has potential for point-of-care nucleic acid testing in the sexual health clinic, has not been assessed for pooled specimen testing. METHODS: We prospectively compared GeneXpert testing of pooled pharyngeal and rectal swabs with urine samples to standard of care testing of individual specimens from 107 participants using the Roche cobas 4800 CT/NG assay (cobas) for CT and NG in high-risk MSM attending an inner city sexual health clinic. RESULTS: We found testing of pooled pharyngeal, rectal and urine samples by the GeneXpert to have 100% agreement for NG and 94% overall agreement for CT when compared with individual specimen testing by cobas. For CT testing, 14 cases were detected for both tests, 4for cobas only, 2 for GeneXpert only and 89 participants were negative for both tests. CONCLUSIONS: Pooled specimen CT and NG testing by the GeneXpert was accurate when compared with single specimen testing and has potential for screening MSM for CT and NG. The role of pooled specimen testing with the GeneXpert as a point-of-care nucleic acid test in MSM requires further investigation.

Diet-induced adipose tissue expansion is mitigated in mice with a targeted inactivation of mesoderm specific transcript (Mest).

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the α/ß-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells.

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.

Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-ß/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

Diet Polyphenol Curcumin Stimulates Hepatic Fgf21 Production and Restores Its Sensitivity in High-Fat-Diet-Fed Male Mice.

We found previously that short-term curcumin gavage stimulated mouse hepatic fibroblast growth factor 21 (Fgf21) expression. Here we conducted mechanistic exploration and investigated the potential pathophysiological relevance on this regulation. Fgf21 stimulation was observed at messenger RNA and protein levels in mice with daily curcumin gavage for 4 or 8 days and in primary hepatocytes with curcumin treatment. Using peroxisome proliferator-activated receptor α (PPARα) agonist and antagonist, along with luciferase reporter and chromatin immune-precipitation approaches, we determined that curcumin stimulates Fgf21 transcription in a mechanism involving PPARα activation. High-fat diet (HFD) feeding also increased mouse hepatic and serum Fgf21 levels, whereas dietary curcumin intervention attenuated these increases. We found that HFD feeding reduced hepatic expression levels of genes that encode FGFR1 and ßKlotho, PGC1α, and the targets of the PPARα-PGC1α axis, whereas concomitant curcumin intervention restored or partially restored their expression levels. Importantly, hepatocytes from HFD-fed mice showed a loss of response to FGF21 treatment on Erk phosphorylation and the expression of Egr1 and cFos; this response was restored in hepatocytes from HFD-fed mice with curcumin intervention. This investigation expanded our mechanistic understanding of the metabolic beneficial effects of dietary curcumin intervention involving the regulation of Fgf21 production and the attenuation of HFD-induced Fgf21 resistance.

Molecular correlates of fat mass expansion in C57BL/6J mice after short-term exposure to dietary fat.

Heterogeneity of obesity within a population of inbred mice fed an obesogenic high-fat diet (HFD) is associated with changes of gene expression in white adipose tissue (WAT). One gene in particular with large variations among mice, mesoderm-specific transcript (Mest), has been shown to be highly inducible after being fed a short-term HFD, and its expression in WAT before HFD feeding is predictive for susceptibility to the development of obesity. To gain further insight into the association of Mest with rapid changes in body composition, 96 individually housed C57BL/6J mice were fed an HFD for only 2 weeks, resulting in a 12-fold and 90-fold variation in Mest mRNA in visceral epididymal and subcutaneous inguinal WAT, respectively. WAT Mest mRNA was positively associated with interindividual variation of fat mass. Surprisingly, there was only a slight association of WAT Mest with food intake when normalized by body weight or lean mass. In addition, WAT Mest expression coincided highly with the expression of the transcription factor Kruppel-like factor 14 (Klf14), an imprinted gene that regulates lipid metabolism in WAT. Our data suggest that KLF14 transcriptional activity may partially mediate, or act in concert with, MEST as part of an epigenetic mechanism that promotes fat mass accumulation in mice fed an obesogenic diet.

The ubiquitin ligase Siah2 regulates obesity-induced adipose tissue inflammation.

OBJECTIVE: Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation was examined. METHODS: Wild-type and Siah2KO mice were fed a low- or high-fat diet for 16 weeks. Indirect calorimetry, body composition, and glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution, and lipolysis were also analyzed. RESULTS: Enlarged adipocytes in obese Siah2KO mice were not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis, and crown-like structures were reduced in the Siah2KO adipose tissue, and Siah2KO adipocytes were more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increased expression of PPARγ target genes involved in lipid metabolism and decreased expression of proinflammatory adipokines regulated by PPARγ. CONCLUSIONS: Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation.

Targeted deletion of FGL2 leads to increased early viral replication and enhanced adaptive immunity in a murine model of acute viral hepatitis caused by LCMV WE.

Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4(+)CD25(+) Foxp3(+) regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2(-/-)) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2(-/-) had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2(+/+)). Frequencies of CD8(+) and CD4(+) T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2(-/-) mice. Increased frequencies of CD8(+) T cells specific for LCMV tetramers GP33 and NP396 were detected within the liver of fgl2(-/-) mice. Plasma from fgl2(-/-) mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2(-/-) mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.

Characterization of fibrinogen-like protein 2 (FGL2): monomeric FGL2 has enhanced immunosuppressive activity in comparison to oligomeric FGL2.

Fibrinogen-like protein 2 (FGL2), a novel effector molecule of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), mediates its suppressive activity through binding to low affinity Fcγ receptors expressed on antigen presenting cells (APCs). FGL2 has been implicated in the pathogenesis of viral hepatitis, xeno- and allotransplant rejection, and rheumatoid arthritis. Here we fully analyzed the structure-function relationships of recombinant murine FGL2 generated in COS-7 cells and identified the receptor binding domains. Native FGL2 exists as an oligomer with a molecular weight of approximately 260 kDa, while under reducing conditions, FGL2 has a molecular weight of 65 kDa suggesting that native FGL2 is composed of four monomers. By site-directed mutation, cysteines at positions 94, 97, 184 and 187, found in the coiled-coil domain were shown to be crucial for FGL2 oligomerization. Monomeric FGL2 had a lower affinity binding to APCs, but increased immunosuppressive activity compared to oligomeric FGL2. Deglycosylation demonstrated that sugar moieties are critical for maintaining solubility of FGL2. SWISS-MODEL analysis suggested that FGL2 has a similar tertiary structure with other members of the fibrinogen family such as fibrinogen and tachylectin. Mutational analysis of cysteine residues and Western blots suggested an asymmetric bouquet-shaped quaternary structure for oligomeric FGL2, resembling many pattern-recognition molecules in the lectin pathway of innate immunity. The functional motifs of FGL2 were mapped to the C terminal globular domain, using a peptide blockade assay. These results collectively define the biochemical and immunological determinants of FGL2, an important immunosuppressive molecule of Treg providing important insights for designing FGL2-related therapeutics.