Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.

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.

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.

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).

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.

Myeloid Cell-Specific IL-4 Receptor Knockout Partially Protects from Adipose Tissue Inflammation.

IL-4 receptor signaling is supposed to play a major role in anti-inflammatory polarization and proliferation of adipose tissue macrophages. In this study, we examined the metabolic and inflammatory phenotype of C57BL/6J mice (IIl4ra) with LysM-dependent knockout (IIl4ra Δmyel) of the IL-4 receptor α-chain (IL-4Rα), the mandatory signaling component of IL-4 and IL-13, on chow and high-fat diet. Lean IIl4ra Δmyel mice showed decreased insulin sensitivity, no divergent adipose tissue macrophage polarization, but an increased percentage of CD8+ T cells in visceral adipose tissue. After 20 wk of a high-fat diet, IIl4ra Δmyel mice exhibited higher glucose tolerance, no changes in the lymphocyte compartment and fewer M1 macrophages in visceral adipose tissue. In vivo adipose tissue macrophage proliferation measured by BrdU incorporation was unaffected by Il4ra knockout. Interestingly, we show that IL-4Rα signaling directly augmented Itgax (Cd11c) gene expression in bone marrow-derived macrophages and increased the amount of CD11c+ macrophages in adipose tissue explants. Myeloid cell-specific knockout of Il4ra deteriorated insulin sensitivity in lean mice but improved parameters of glucose homeostasis and partially protected from adipose tissue inflammation in obese mice. Hence, IL-4Rα signaling probably plays a minor role in maintaining the macrophage M2 population and proliferation rates in vivo. Moreover, our data indicate that IL-4 signaling plays a proinflammatory role in adipose tissue inflammation by directly upregulating CD11c on adipose tissue macrophages.

Genetic amniocentesis using atraumatic 29 gauge needle in patients having a chorioamniotic separation.

OBJECTIVES: Chorioamniotic separation (CAS) at the time of standard amniocentesis (AC) is a risk factor for postprocedural complications and should be avoided. The aim of this study was to quantify procedure-related risks after AC with a 29G-needle in cases of CAS, and evaluation of perinatal outcome in CAS after 15 weeks' gestation (GW). METHODS: Retrospective analysis of genetic AC with a pencil-point 29G needle after 15 completed GW in pregnancies, in which the fetal membranes were not yet fused. Included into the study were women aged 16-44 years with at least 15 completed GWs referred for second trimester AC to identify fetal chromosomal aberrations. RESULTS: 437 ACs were made in total with the 29G-needle. The median maternal age was 30 (16-44) years. 145 cases showed CAS where the distance between chorion and amnion was 0.10-10.02 mm at AC. 38 pregnancies were terminated, 37 of which had a genetic disorder. The risk of aneuploidy increases by a factor of 2 (95% CI 1.4-2.8) for every 1 mm of CAS enlargement. No procedure-related complications were found up to two weeks after the AC. CONCLUSIONS: CAS seems to be massively underreported. Early diagnosis in case of CAS is something to strive for as CAS could be an indicator of genetic abnormalities - a "soft marker". With the atraumatic 29G needle, the risk of complications after AC in CAS seems to be very low.

The Role of IL-13 and IL-4 in Adipose Tissue Fibrosis.

White adipose tissue (WAT) fibrosis, characterized by an excess of extracellular (ECM) matrix components, is strongly associated with WAT inflammation and dysfunction due to obesity. Interleukin (IL)-13 and IL-4 were recently identified as critical mediators in the pathogenesis of fibrotic diseases. However, their role in WAT fibrosis is still ill-defined. We therefore established an ex vivo WAT organotypic culture system and demonstrated an upregulation of fibrosis-related genes and an increase of α-smooth muscle actin (αSMA) and fibronectin abundance upon dose-dependent stimulation with IL-13/IL-4. These fibrotic effects were lost in WAT lacking il4ra, which encodes for the underlying receptor controlling this process. Adipose tissue macrophages were found to play a key role in mediating IL-13/IL-4 effects in WAT fibrosis as their depletion through clodronate dramatically decreased the fibrotic phenotype. IL-4-induced WAT fibrosis was partly confirmed in mice injected intraperitoneally with IL-4. Furthermore, gene correlation analyses of human WAT samples revealed a strong positive correlation of fibrosis markers with IL-13/IL-4 receptors, whereas IL13 and IL4 correlations failed to confirm this association. In conclusion, IL-13 and IL-4 can induce WAT fibrosis ex vivo and partly in vivo, but their role in human WAT remains to be further elucidated.

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.

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.

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.

Treatment-Induced Neuropathy in Diabetes (TIND)-Developing a Disease Model in Type 1 Diabetic Rats.

Treatment-induced neuropathy in diabetes (TIND) is defined by the occurrence of an acute neuropathy within 8 weeks of an abrupt decrease in glycated hemoglobin-A1c (HbA1c). The underlying pathogenic mechanisms are still incompletely understood with only one mouse model being explored to date. The aim of this study was to further explore the hypothesis that an abrupt insulin-induced fall in HbA1c may be the prime causal factor of developing TIND. BB/OKL (bio breeding/OKL, Ottawa Karlsburg Leipzig) diabetic rats were randomized in three groups, receiving insulin treatment by implanted subcutaneous osmotic insulin pumps for 3 months, as follows: Group one received 2 units per day; group two 1 unit per day: and group three 1 unit per day in the first month, followed by 2 units per day in the last two months. We serially examined blood glucose and HbA1c levels, motor- and sensory/mixed afferent conduction velocities (mNCV and csNCV) and peripheral nerve morphology, including intraepidermal nerve fiber density and numbers of Iba-1 (ionized calcium binding adaptor molecule 1) positive macrophages in the sciatic nerve. Only in BB/OKL rats of group three, with a rapid decrease in HbA1c of more than 2%, did we find a significant decrease in mNCV in sciatic nerves (81% of initial values) after three months of treatment as compared to those group three rats with a less marked decrease in HbA1c <2% (mNCV 106% of initial values, p ≤ 0.01). A similar trend was observed for sensory/mixed afferent nerve conduction velocities: csNCV were reduced in BB/OKL rats with a rapid decrease in HbA1c >2% (csNCV 90% of initial values), compared to those rats with a mild decrease <2% (csNCV 112% of initial values, p ≤ 0.01). Moreover, BB/OKL rats of group three with a decrease in HbA1c >2% showed significantly greater infiltration of macrophages by about 50% (p ≤ 0.01) and a decreased amount of calcitonin gene related peptide (CGRP) positive nerve fibers as compared to the animals with a milder decrease in HbA1c. We conclude that a mild acute neuropathy with inflammatory components was induced in BB/OKL rats as a consequence of an abrupt decrease in HbA1c caused by high-dose insulin treatment. This experimentally induced neuropathy shares some features with TIND in humans and may be further explored in studies into the pathogenesis and treatment of TIND.

Reactive Nanoparticles Derived from Polysaccharide Phenyl Carbonates.

Polysaccharide (PS) based nanoparticles (NP) are of great interest for biomedical applications. A key challenge in this regard is the functionalization of these nanomaterials. The aim of the present work was the development of reactive PS-NP that can be coupled with an amino group containing compounds under mild aqueous conditions. A series of cellulose phenyl carbonates (CPC) and xylan phenyl carbonates (XPC) with variable degrees of substitution (DS) was obtained by homogeneous synthesis. The preparation of PS-NP by self-assembling of these hydrophobic derivatives was studied comprehensively. While CPC mostly formed macroscopic aggregates, XPC formed well-defined spherical NP with diameters around 100 to 200 nm that showed a pronounced long-term stability in water against both particle aggregation as well as cleavage of phenyl carbonate moieties. Using an amino group functionalized dye it was demonstrated that the novel XPC-NP are reactive towards amines. A simple coupling procedure was established that enables direct functionalization of the reactive NP in an aqueous dispersion. Finally, it was demonstrated that dye functionalized XPC-NP are non-cytotoxic and can be employed in advanced biomedical applications.

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.

The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional relevance.

BACKGROUND: G protein-coupled receptors (GPCR) are well-characterized regulators of a plethora of physiological functions among them the modulation of adipogenesis and adipocyte function. The class of Adhesion GPCR (aGPCR) and their role in adipose tissue, however, is poorly studied. With respect to the demand for novel targets in obesity treatment, we present a comprehensive study on the expression and function of this enigmatic GPCR class during adipogenesis and in mature adipocytes. METHODS: The expression of all aGPCR representatives was determined by reanalyzing RNA-Seq data and by performing qPCR in different mouse and human adipose tissues under low- and high-fat conditions. The impact of aGPCR expression on adipocyte differentiation and lipid accumulation was studied by siRNA-mediated knockdown of all expressed members of this receptor class. The biological characteristics and function of mature adipocytes lacking selected aGPCR were analyzed by mass spectrometry and biochemical methods (lipolysis, glucose uptake, adiponectin secretion). RESULTS: More than ten aGPCR are significantly expressed in visceral and subcutaneous adipose tissues and several aGPCR are differentially regulated under high-caloric conditions in human and mouse. Receptor knockdown of six receptors resulted in an impaired adipogenesis indicating their expression is essential for proper adipogenesis. The altered lipid composition was studied in more detail for two representatives, ADGRG2/GPR64 and ADGRG6/GPR126. While GPR126 is mainly involved in adipocyte differentiation, GPR64 has an additional role in mature adipocytes by regulating metabolic processes. CONCLUSIONS: Adhesion GPCR are significantly involved in qualitative and quantitative adipocyte lipid accumulation and can control lipolysis. Factors driving adipocyte formation and function are governed by signaling pathways induced by aGPCR yielding these receptors potential targets for treating obesity.

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.

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.

Intestinal nerve cell injury occurs prior to insulin resistance in female mice ingesting a high-fat diet.

Diabetic patients suffer from gastrointestinal disorders associated with dysmotility, enteric neuropathy and dysbiosis of gut microbiota; however, gender differences are not fully known. Previous studies have shown that a high-fat diet (HFD) causes type two diabetes (T2D) in male mice after 4-8 weeks but only does so in female mice after 16 weeks. This study seeks to determine whether sex influences the development of intestinal dysmotility, enteric neuropathy and dysbiosis in mice fed HFD. We fed 8-week-old C57BL6 male and female mice a standard chow diet (SCD) or a 72% kcal HFD for 8 weeks. We analyzed the associations between sex and intestinal dysmotility, neuropathy and dysbiosis using motility assays, immunohistochemistry and next-generation sequencing. HFD ingestion caused obesity, glucose intolerance and insulin resistance in male but not female mice. However, HFD ingestion slowed intestinal propulsive motility in both male and female mice. This was associated with decreased inhibitory neuromuscular transmission, loss of myenteric inhibitory motor neurons and axonal swelling and loss of cytoskeletal filaments. HFD induced dysbiosis and changed the abundance of specific bacteria, especially Allobaculum, Bifidobacterium and Lactobacillus, which correlated with dysmotility and neuropathy. Female mice had higher immunoreactivity and numbers of myenteric inhibitory motor neurons, matching larger amplitudes of inhibitory junction potentials. This study suggests that sex influences the development of HFD-induced metabolic syndrome but dysmotility, neuropathy and dysbiosis occur independent of sex and prior to T2D conditions. Gastrointestinal dysmotility, neuropathy and dysbiosis might play a crucial role in the pathophysiology of T2D in humans irrespective of sex.

IL-6 Regulates M2 Polarization and Local Proliferation of Adipose Tissue Macrophages in Obesity.

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 focal sites of inflammation around dying adipocytes, so-called crown-like structures, exhibit a unique microenvironment for macrophage proliferation. Interestingly, locally proliferating macrophages were not classically activated (M1), but they exhibited a rather alternatively activated (M2) immune phenotype. In this study, we established organotypic cell cultures of AT explants to study the impact of cytokine treatment on local ATM proliferation, without the bias of early monocyte recruitment. We show that exposure of AT to Th2 cytokines, such as IL-4, IL-13, and GM-CSF, stimulates ATM proliferation, whereas Th1 cytokines, such as TNF-α, inhibit local ATM proliferation. Furthermore, AT from obese mice exhibits an increased sensitivity to IL-4 stimulation, indicated by an increased phosphorylation of STAT6. In line with this, gene expression of the IL-4 receptor (Il4ra) and its ligand IL-13 are elevated in AT of obese C57BL/6 mice. Most importantly, Il4ra expression and susceptibility to IL-4 or IL-13 treatment depend on IL-6 signaling, which seems to be the underlying mechanism of local ATM proliferation in obesity. We conclude that IL-6 acts as a Th2 cytokine in obesity by stimulating M2 polarization and local ATM proliferation, presumably due to upregulation of the IL-4 receptor α.

Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high fat diet-induced obesity by counteracting adipose tissue inflammation in vivo.

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 -/-) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 -/- mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 -/-. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.