Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

IL-6 signaling drives self-renewal and alternative activation of adipose tissue macrophages.

Introduction: Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that neutralization of interleukin (IL)-6 in obese AT organ cultures inhibits proliferation of ATMs, which occurs preferentially in alternatively activated macrophage phenotype. Methods: In this study, we investigated AT biology and the metabolic phenotype of mice with myeloid cell-specific IL-6Rα deficiency (Il6ra Δmyel) after normal chow and 20 weeks of high-fat diet focusing on AT inflammation, ATM polarization and proliferation. Using organotypical AT culture and bone marrow derived macrophages (BMDMs) of IL-4Rα knockout mice (Il4ra -/-) we studied IL-6 signaling. Results: Obese Il6ra Δmyel mice exhibited no differences in insulin sensitivity or histological markers of AT inflammation. Notably, we found a reduction of ATMs expressing the mannose receptor 1 (CD206), as well as a decrease of the proliferation marker Ki67 in ATMs of Il6ra Δmyel mice. Importantly, organotypical AT culture and BMDM data of Il4ra -/- mice revealed that IL-6 mediates a shift towards the M2 phenotype independent from the IL-6/IL-4Rα axis. Discussion: Our results demonstrate IL-4Rα-independent anti-inflammatory effects of IL-6 on macrophages and the ability of IL-6 to maintain proliferation rates in obese AT.

Mixed reality for spine surgery: a step into the future with a human cadaveric accuracy study.

OBJECTIVE: Current application of mixed reality as a navigation aid in the field of spinal navigation points to the potential of this technology in spine surgery. Crucial factors for acceptance include intuitive workflow, system stability, reliability, and accuracy of the method. The authors therefore aimed to investigate the accuracy of the system in visualization of anatomical structures using mixed reality in the example of pedicles of the thoracic spine in a human cadaveric study. Potential difficulties and limitations are discussed. METHODS: CT scans of a human cadaveric spinal column specimen were performed. After segmentation and import into the advanced HoloLens 2 software, the vertebrae were exposed. The vertebral arches were preserved on one side for a landmark-based surface registration, whereas pedicles were exposed on the other side in order to measure and evaluate deviation of the overlay holographs with regard to the exact anatomical structure. Accuracy was measured and statistically evaluated. RESULTS: In this work it was demonstrated that the overlay of the virtual 3D model pedicles with the real anatomical structures with anatomical landmark registration was within an acceptable surgical accuracy with the mean value of 2.1 mm (maximum 3.8 mm, minimum 1.2 mm). The highest accuracy was registered at the medial and lateral pedicle wall, and the measurement results were best in the region of the middle thoracic spine. CONCLUSIONS: The accuracy analysis for mixed reality (i.e., between the virtual and real anatomical situation of the thoracic spine) showed a very good agreement when focus was on the pedicles. This work is thus a rare proof of the precision of segmentation to the potential surgical area. The results encourage researchers to open up mixed reality technology in its development and application for spinal navigation.

Direct Functionalization of Polysaccharide-Based Xylan Phenyl Carbonate Nanoparticles with Tumor Cell Specific Antibodies.

An efficient and easy-to-use approach is presented for obtaining biocompatible polysaccharide-based nanoparticles (NP) that can act as tumor-specific drug delivery agents. Two antibodies are directly immobilized onto reactive xylan phenyl carbonate (XPC) NP; namely Cetuximab (CTX) that binds to human epidermal growth factor receptor (EGFR) and Atezolizumab (ATZ) that binds to programmed death-ligand 1 (PD-L1). High coupling efficiency (up to 100 %) are achieved without any pre-activation and no aggregation occurs during antibody immobilization. By quartz crystal microbalance experiments with dissipation monitoring (QCM-D), flow cytometry assays, and confocal laser scanning microscopy imaging it is demonstrated that the functionalized XPC-NP specifically bind to cells carrying the corresponding antigens. Moreover, the NP retain the antibody specific bioactivities (growth inhibition for CTX and induction of T-cell cytotoxicity for ATZ).

Adipose tissue macrophage dysfunction is associated with a breach of vascular integrity in NASH.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. METHODS: Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. RESULTS: We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. CONCLUSIONS: NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. IMPACT AND IMPLICATIONS: Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.

Macrophages in obesity are characterised by increased IL-1ß response to calcium-sensing receptor signals.

OBJECTIVE: Obesity is complicated by inflammatory activation of the innate immune system. Stimulation of the calcium-sensing receptor (CaSR) by extra-cellular calcium ions ([Ca2+]ex) can trigger NLRP3 inflammasome activation and inflammation. We hypothesised, that this mechanism might contribute to the activation of adipose tissue (AT) in obesity, and investigated [Ca2+]ex-induced, CaSR mediated IL-1ß release by macrophages in obesity. METHODS: [Ca2+]ex-induced IL-1ß release was investigated in monocyte-derived macrophages (MDM) generated from peripheral blood of patients with obesity and from normal-weight controls. Visceral and subcutaneous AT biosamples were stimulated with [Ca2+]ex, and IL-1ß release, as well as expression of NLRP3 inflammasome and cytokine genes, was determined. RESULTS: Both MDM and AT readily responded with concentration-dependent IL-1ß release already at low, near physiological concentrations to addition of [Ca2+]ex, which was more than 80 fold higher than the LPS-induced effect. IL-1ß levels induced by [Ca2+]ex were significantly higher not only in MDM from patients with obesity compared to controls, but also in visceral versus subcutaneous AT. This fat-depot difference was also reflected by mRNA expression levels of inflammasome and cytokine genes. CONCLUSIONS: Obesity renders macrophages more susceptible to [Ca2+]ex-induced IL-1ß release and pyroptosis. Increased susceptibility was independent of the response to LPS and circulating CRP arguing against mere pro-inflammatory pre-activation of monocytes. Instead, we propose that CaSR mediated signalling is relevant for the deleterious innate immune activation in obesity.

Multiomics reveal unique signatures of human epiploic adipose tissue related to systemic insulin resistance.

OBJECTIVE: Human white adipose tissue (AT) is a metabolically active organ with distinct depot-specific functions. Despite their locations close to the gastrointestinal tract, mesenteric AT and epiploic AT (epiAT) have only scarcely been investigated. Here, we aim to characterise these ATs in-depth and estimate their contribution to alterations in whole-body metabolism. DESIGN: Mesenteric, epiploic, omental and abdominal subcutaneous ATs were collected from 70 patients with obesity undergoing Roux-en-Y gastric bypass surgery. The metabolically well-characterised cohort included nine subjects with insulin sensitive (IS) obesity, whose AT samples were analysed in a multiomics approach, including methylome, transcriptome and proteome along with samples from subjects with insulin resistance (IR) matched for age, sex and body mass index (n=9). Findings implying differences between AT depots in these subgroups were validated in the entire cohort (n=70) by quantitative real-time PCR. RESULTS: While mesenteric AT exhibited signatures similar to those found in the omental depot, epiAT was distinct from all other studied fat depots. Multiomics allowed clear discrimination between the IS and IR states in all tissues. The highest discriminatory power between IS and IR was seen in epiAT, where profound differences in the regulation of developmental, metabolic and inflammatory pathways were observed. Gene expression levels of key molecules involved in AT function, metabolic homeostasis and inflammation revealed significant depot-specific differences with epiAT showing the highest expression levels. CONCLUSION: Multi-omics epiAT signatures reflect systemic IR and obesity subphenotypes distinct from other fat depots. Our data suggest a previously unrecognised role of human epiploic fat in the context of obesity, impaired insulin sensitivity and related diseases.

Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages.

A chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.

CD4+ T cells regulate glucose homeostasis independent of adipose tissue dysfunction in mice.

Obesity is frequently associated with a chronic low-grade inflammation in the adipose tissue (AT) and impaired glucose homeostasis. Adipose tissue macrophages (ATMs) have been shown to accumulate in the inflamed AT either by means of recruitment from the blood or local proliferation. ATM proliferation and activation can be stimulated by TH2 cytokines, such as IL-4 and IL-13, suggesting involvement of CD4-positive T cells in ATM proliferation and activation. Furthermore, several studies have associated T cells to alterations in glucose metabolism. Therefore, we sought to examine a direct impact of CD4-positive T cells on ATM activation, ATM proliferation and glucose homeostasis using an in vivo depletion model. Surprisingly, CD4 depletion did not affect ATM activation, ATM proliferation, or insulin sensitivity. However, CD4 depletion led to a significant improvement of glucose tolerance. In line with this, we found moderate disturbances in pancreatic endocrine function following CD4 depletion. Hence, our data suggest that the effect on glucose metabolism observed after CD4 depletion might be mediated by organs other than AT and independent of AT inflammation.

Evaluating Release Kinetics from Alginate Beads Coated with Polyelectrolyte Layers for Sustained Drug Delivery.

Current approaches in stem cell-based bone tissue engineering require a release of bioactive compounds over up to 2 weeks. This study presents a polyelectrolyte-layered system featuring sustained release of water-soluble drugs with decreased burst release. The bioactive compounds adenosine 5'-triphosphate (ATP), suramin, and A740003 (a less water-soluble purinergic receptor ligand) were incorporated into alginate hydrogel beads subsequently layered with different polyelectrolytes (chitosan, poly(allyl amine), alginate, or lignosulfonate). Drug release into aqueous medium was monitored over 14 days and evaluated using Korsmeyer-Peppas, Peppas-Sahlin, Weibull models, and a Langmuir-like "Two-Stage" model. Release kinetics strongly depended on both the drug and the polyelectrolyte system. For ATP, five alternating layers of poly(allyl amine) and alginate proved to be most effective in sustaining the release. Release of suramin could be prolonged best with lignosulfonate as polyanion. A740003 showed prolonged release even without layering. Applying polyelectrolyte layers significantly slowed down the burst release. Release curves could be best described with the Langmuir-like model.

Expression of the EWSR1-FLI1 fusion oncogene in pancreas cells drives pancreatic atrophy and lipomatosis.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) harbors mutant KRAS as the most common driver mutation. Studies on mouse models have uncovered the tumorigenic characteristics of the Kras oncogene driving pancreatic carcinogenesis. Similarly, Ewing sarcoma predominantly depends on the occurrence of the EWSR1-FLI1 fusion oncogene. The expression of EWSR1-FLI1 affects pro-tumorigenic pathways and induces cell transformation. In this study, we investigated whether mutant Kras could be exchanged by another potent oncogene, such as EWSR1-FLI1, to initiate pancreatic cancer development. METHODS: We generated two conditional mouse models expressing mutant KrasG12D (KC) or the EWSR1-FLI1 oncogene (E/F) in pancreas cells. Pancreatic tissue was collected from the mice at 4-6 weeks and 11-13 weeks of age as well as from survival cohorts to determine the development of spontaneous acinar-to-ductal metaplasia (ADM) and neoplastic lesions. Immunohistochemistry and immunofluorescence staining were performed to characterize and quantify changes in tissue morphology. RESULTS: The expression of the EWSR1-FLI1 fusion protein in pancreas cells was confirmed by positive FLI1 immunohistochemistry staining. Notably, the EWSR1-FLI1 expression in pancreas cells resulted in a strong depletion of the acinar cell mass and an extensive lipomatosis. Although the E/F mice exhibited spontaneous ADM formation and a shorter overall survival rate compared to KC mice, no development of neoplastic lesion was observed in aging E/F mice. CONCLUSIONS: The expression of the EWSR1-FLI1 oncogene leads to a strong pancreatic atrophy and lipomatosis. ADM formation indicates that pancreatic acinar cells are susceptible for EWSR1-FLI1-mediated oncogenic transformation to a limited extent. However, the EWSR1-FLI1 oncogene is insufficient to induce pancreatic cancer development.

Examination of ex-vivo viability of human adipose tissue slice culture.

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 µm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.

Adipose tissue derived bacteria are associated with inflammation in obesity and type 2 diabetes.

OBJECTIVE: Bacterial translocation to various organs including human adipose tissue (AT) due to increased intestinal permeability remains poorly understood. We hypothesised that: (1) bacterial presence is highly tissue specific and (2) related in composition and quantity to immune inflammatory and metabolic burden. DESIGN: We quantified and sequenced the bacterial 16S rRNA gene in blood and AT samples (omental, mesenteric and subcutaneous) of 75 subjects with obesity with or without type 2 diabetes (T2D) and used catalysed reporter deposition (CARD) - fluorescence in situ hybridisation (FISH) to detect bacteria in AT. RESULTS: Under stringent experimental and bioinformatic control for contaminants, bacterial DNA was detected in blood and omental, subcutaneous and mesenteric AT samples in the range of 0.1 to 5 pg/µg DNA isolate. Moreover, CARD-FISH allowed the detection of living, AT-borne bacteria. Proteobacteria and Firmicutes were the predominant phyla, and bacterial quantity was associated with immune cell infiltration, inflammatory and metabolic parameters in a tissue-specific manner. Bacterial composition differed between subjects with and without T2D and was associated with related clinical measures, including systemic and tissues-specific inflammatory markers. Finally, treatment of adipocytes with bacterial DNA in vitro stimulated the expression of TNFA and IL6. CONCLUSIONS: Our study provides contaminant aware evidence for the presence of bacteria and bacterial DNA in several ATs in obesity and T2D and suggests an important role of bacteria in initiating and sustaining local AT subclinical inflammation and therefore impacting metabolic sequelae of obesity.

Unspecific DNA recombination in AdipoqCre-ERT2 - mediated knockout approaches in transgenic mice is sex-, age- and genotype-dependent.

Due to the epidemic rise of obesity prevalence, adipose tissue (AT) research is of major interest. Our aim was to study specificity of the most-common Cre/loxP approach for inducible gene manipulation of AT in mice (AdipoqCre-ERT2). We used mice with tamoxifen-sensitive Cre recombinase controlled by the adiponectin promoter (AdipoqCre-ERT2), which were crossed to a tdTomato reporter mouse to visualize the site of recombination on a single-cell resolution. Albeit tamoxifen induced tdTomato expression in this model, also non-stimulated background recombination ('Cre leakage') was detected in AT of untreated Adipoq-CreERT2xTDTO mice in vivo. Quantification of Cre leakage revealed age, sex and genotype as factors impacting on non-induced Cre recombination.

Autophagy differentially regulates macrophage lipid handling depending on the lipid substrate (oleic acid vs. acetylated-LDL) and inflammatory activation state.

The regulation of lipid droplet (LD) dynamics by autophagy in naïve macrophages is complex: Inhibiting autophagosome initiation steps attenuates oleic acid (OA) induced LD (OA-LD) biogenesis, whereas interfering with later-autophagosome maturation/lysosomal steps accelerates OA-LD biogenesis rate, but not OA-LD degradation. Here we hypothesized that regulation of macrophage lipid handling by autophagy may be lipid-substrate and activation-state-specific. Using automated quantitative live-cell imaging, initial LD biogenesis rate was ~30% slower when the lipid source was acetylated low density lipoprotein (acLDL) compared to OA. Yet, both were similarly affected by triacsin-C, an inhibitor of acyl-CoA synthase, which inhibited, and etomoxir, an inhibitor of acylcarnitine palmitoyl transferase (fatty acid oxidation), which augmented, LD biogenesis rates. An autophagy inducing peptide, Tat-Beclin1, enhanced the degradation, and inhibited (by 37%) the biogenesis of acLDL induced LD (acLDL-LD). Yet, Tat-Beclin1 increased OA-LD biogenesis rate by 70%. When macrophages were pre-activated with LPS + INFG they exhibited increased autophagosome number and area, and reduced BECN1 and ATG14 protein levels, which associated with a markedly attenuated autophagic flux. Concomitantly, OA-LD and acLDL-LD biogenesis rates increased 3 and 7.4-fold, respectively, but could not be further modulated by Tat-Beclin1, as observed in non-activated/naïve macrophages. We propose that macrophage autophagy, and/or components of its machinery, differentially regulate LD/foam-cell biogenesis depending on the lipid-source, and that inflammatory activation uncouples autophagy from LD biogenesis.

Non-Cytotoxic Agarose/Hydroxyapatite Composite Scaffolds for Drug Release.

Healing of large bone defects requires implants or scaffolds that provide structural guidance for cell growth, differentiation, and vascularization. In the present work, an agarose-hydroxyapatite composite scaffold was developed that acts not only as a 3D matrix, but also as a release system. Hydroxyapatite (HA) was incorporated into the agarose gels in situ in various ratios by a simple procedure consisting of precipitation, cooling, washing, and drying. The resulting gels were characterized regarding composition, porosity, mechanical properties, and biocompatibility. A pure phase of carbonated HA was identified in the scaffolds, which had pore sizes of up to several hundred micrometers. Mechanical testing revealed elastic moduli of up to 2.8 MPa for lyophilized composites. MTT testing on Lw35human mesenchymal stem cells (hMSCs) and osteosarcoma MG-63 cells proved the biocompatibility of the scaffolds. Furthermore, scaffolds were loaded with model drug compounds for guided hMSC differentiation. Different release kinetic models were evaluated for adenosine 5'-triphosphate (ATP) and suramin, and data showed a sustained release behavior over four days.

Impact of body weight gain on hepatic metabolism and hepatic inflammatory cytokines in comparison of Shetland pony geldings and Warmblood horse geldings.

BACKGROUND: Non-alcoholic fatty liver disease is known as determining part of human obesity. The impact of body weight (BW) gain on liver metabolism has not been extensively investigated yet. OBJECTIVES: To investigate hepatic alterations caused by increasing BW in ponies and horses. ANIMALS: A total of 19 non-obese equines (10 Shetland ponies, geldings; nine Warmblood horses, geldings). METHODS: Animals received 200% of their metabolizable maintenance energy requirements for 2 years. Serum alkaline phosphatase, glutamate dehydrogenase (GLDH), aspartate aminotransferase (AST), and gamma-glutamyl transferase activities and bile acids were analyzed several times during 2 years of hypercaloric diet. Hepatic lipid content and hepatic levels of the interleukin (IL)-6, tumor necrosis factor α (TNFα), cluster of differentiation (CD) 68, IL-1ß, lipoprotein lipase (LPL), fatty acid-binding protein 1, chemerin and nuclear factor-κB mRNAs were assessed at the start of the study and after 1 and 2 years of excess energy intake. RESULTS: The mean (±SD) BW gain recorded during 2 years of excess energy intake was 29.9 ± 19.4% for ponies and 17 ± 6.74% for horses. The hepatic lipid content was not profoundly affected by increasing BW. Levels of the IL-6, TNFα, CD68 and IL-1ß mRNAs did not change during BW gain. Levels of the chemerin mRNA increased significantly in both breeds (ponies: P = 0.02; horses: P = 0.02) in response to BW gain. Significant differences in serum GLDH and AST activities, serum bile acid concentrations and hepatic levels of the LPL mRNA were observed between ponies and horses at the end of the study. CONCLUSIONS: Chemerin might represent an interesting marker for future equine obesity research. Interestingly, steatosis caused by increasing BW may occur later in the development of obesity in equines than in humans. Additionally, the hepatic metabolism exhibits differences between ponies and horses, which may explain in part the greater susceptibility of ponies to obesity-associated metabolic dysregulations.

Leptin decreases circulating inflammatory IL-6 and MCP-1 in mice.

Leptin influences inflammation and immune response. Dose-dependent effects of leptin on biomarkers of inflammation have not been studied in vivo, so far. Leptin-deficient low-density lipoprotein receptor (LDLR) knockout (LDLR-/- ;ob/ob) female mice were treated with three different leptin doses or saline for 12 weeks. The effect of leptin on plasma interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 concentrations and Il-6 and Mcp-1 mRNA expression in vivo were assessed. Macrophage infiltration in epididymal adipose tissue (epiAT) after leptin treatment was determined by quantitative immunohistochemical analysis. Aortic root atherosclerotic lesions were analyzed by oil red O staining. Mean plasma IL-6 and MCP-1 decreased significantly in the 3.0 mg/kg BW/day group as compared to control mice (both P < 0.01). Messenger RNA expression of Il-6 and Mcp-1 was significantly down-regulated by leptin treatment in different adipose tissues in vivo. Characteristic crown-like structures formed by adipose tissue macrophages were significantly reduced by leptin treatment in epiAT. Recombinant leptin dose-dependently diminished plaque area in the aortic root. Leptin administration within the subphysiological to physiological range diminishes circulating pro-inflammatory IL-6 and MCP-1. Reduction of Il-6 and Mcp-1 gene expression in adipose tissue, as well as decreased adipose tissue macrophage infiltration might contribute. © 2018 BioFactors, 45(1):43-48, 2019.

Tribbles homolog 1 deficiency modulates function and polarization of murine bone marrow-derived macrophages.

Macrophages are essential for innate immunity and inflammatory responses and differentiate into various functional phenotypes. Tribbles homolog 1 (Trib1), a member of the mammalian Tribbles homolog pseudokinase family, has been implicated in regulation of cell differentiation, proliferation, and metabolism, but its role in macrophage biology has not been fully elucidated. Here, we investigated the consequences of Trib1 deficiency on macrophage functions and M1/M2 polarization. Bone marrow-derived macrophages (BMDMs) from Trib1-deficient (Trib1-/-) mice exhibited elevated phagocytic capacity, correlating with up-regulation of several scavenger receptors. Concomitantly, uptake of modified low-density lipoprotein was increased in Trib1-/- BMDMs. Trib1-/- macrophages also exhibited diminished migration in the presence of the chemokine MCP-1, associated with reduced expression of the MCP-1 receptor Ccr2 Furthermore, Trib1 deficiency attenuated the response of BMDMs to both M1 and M2 stimuli; induction of the M1-marker genes Il6, Il1b, and Nos2 upon LPS/IFNγ stimulation and of the M2-marker genes Cd206, Fizz1, and Arg1 upon IL-4 stimulation was reduced. Functionally, Trib1 deficiency decreased secretion of proinflammatory cytokines (IL-6, TNFα, IL-1ß, and CXCL1) and reduced nitric oxide and reactive oxygen species production in M1-polarized macrophages. Supporting the attenuated M2 phenotype, IL-4-stimulated Trib1-/- macrophages secreted less IL-10 and TGFß. Mechanistically, Trib1-/- BMDMs displayed lower levels of Janus kinase 1 (JAK1), resulting in reduced activation of LPS/IFNγ-mediated STAT1 signaling. Likewise, decreased levels of JAK1 along with lower activation of STAT6 and STAT3 were observed in M2-polarized Trib1-/- BMDMs. Our findings suggest that Trib1 extensively controls macrophage M1/M2 polarization via the JAK/STAT signaling pathway.

Chitosan-Cellulose Multifunctional Hydrogel Beads: Design, Characterization and Evaluation of Cytocompatibility with Breast Adenocarcinoma and Osteoblast Cells.

Cytocompatible polysaccharide-based functional scaffolds are potential extracellular matrix candidates for soft and hard tissue engineering. This paper describes a facile approach to design cytocompatible, non-toxic, and multifunctional chitosan-cellulose based hydrogel beads utilising polysaccharide dissolution in sodium hydroxide-urea-water solvent system and coagulation under three different acidic conditions, namely 2 M acetic acid, 2 M hydrochloric acid, and 2 M sulfuric acid. The effect of coagulating medium on the final chemical composition of the hydrogel beads is investigated by spectroscopic techniques (ATR-FTIR, Raman, NMR), and elemental analysis. The beads coagulated in 2 M acetic acid displayed an unchanged chitosan composition with free amino groups, while the beads coagulated in 2 M hydrochloric and sulfuric acid showed protonation of amino groups and ionic interaction with the counterions. The ultrastructural morphological study of lyophilized beads showed that increased chitosan content enhanced the porosity of the hydrogel beads. Furthermore, cytocompatibility evaluation of the hydrogel beads with human breast adenocarcinoma cells (soft tissue) showed that the beads coagulated in 2 M acetic acid are the most suitable for this type of cells in comparison to other coagulating systems. The acetic acid fabricated hydrogel beads also support osteoblast growth and adhesion over 192 h. Thus, in future, these hydrogel beads can be tested in the in vitro studies related to breast cancer and for bone regeneration.