Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

Cellular and Molecular Probing of Intact Human Organs.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

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.

Origin, fate and dynamics of macrophages at central nervous system interfaces.

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Sickle hemoglobin confers tolerance to Plasmodium infection.

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

Central Nervous System Stromal Cells Control Local CD8(+) T Cell Responses during Virus-Induced Neuroinflammation.

Stromal cells generate a complex cellular scaffold that provides specialized microenvironments for lymphocyte activation in secondary lymphoid organs. Here, we assessed whether local activation of stromal cells in the central nervous system (CNS) is mandatory to transfer immune recognition from secondary lymphoid organs into the infected tissue. We report that neurotropic virus infection in mice triggered the establishment of such stromal cell niches in the CNS. CNS stromal cell activation was dominated by a rapid and vigorous production of CC-motif chemokine receptor (CCR) 7 ligands CCL19 and CCL21 by vascular endothelial cells and adjacent fibroblastic reticular cell (FRC)-like cells in the perivascular space. Moreover, CCR7 ligands produced by CNS stromal cells were crucial to support recruitment and local re-activation of antiviral CD8(+) T cells and to protect the host from lethal neuroinflammatory disease, indicating that CNS stromal cells generate confined microenvironments that control protective T cell immunity.

Is microglial dystrophy a form of cellular senescence? An analysis of senescence markers in the aged human brain.

Aging can cause morphological transformation in human microglia indicative of cell senescence, termed microglial dystrophy. However, cellular senescence is characterized by additional changes, such as an irregular cell cycle arrest, and a variety of metabolic and molecular changes including a senescence-associated secretory phenotype, dysfunction of degradation mechanisms, and altered DNA damage response. Here, we tested whether dystrophic microglia display customary markers of cell senescence by performing double and triple staining in sections of the temporal lobe and brain stem from 14 humans. We found that markers related to oxidative damage, such as upregulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), hemeoxygenase-1 (HO-1), and y-H2AX, as well as inclusion of lipofuscin, do not or only exceptionally colocalize with dystrophic microglia. Further, we did not observe a decline in lamin B1 around nuclear laminae in either dystrophic or ramified microglia within the same microscopic field. Only ferritin expression, which is known to increase with aging in CNS microglia, was frequently observed in dystrophic, but rarely in ramified microglial cells. We conclude that neither dystrophic nor ramified microglia in human brain exhibit significant expression of conventional senescence markers associated with oxidative stress, and that ferritin is the dominant immunophenotypic change related to microglial aging. We suggest that multiple pathogenic mechanisms other than those driving cellular senescence contribute to dystrophic transformation of microglia.

IGF1R expression by adult oligodendrocytes is not required in the steady-state but supports neuroinflammation.

In the central nervous system (CNS), insulin-like growth factor 1 (IGF-1) regulates myelination by oligodendrocyte (ODC) precursor cells and shows anti-apoptotic properties in neuronal cells in different in vitro and in vivo systems. Previous work also suggests that IGF-1 protects ODCs from cell death and enhances remyelination in models of toxin-induced and autoimmune demyelination. However, since evidence remains controversial, the therapeutic potential of IGF-1 in demyelinating CNS conditions is unclear. To finally shed light on the function of IGF1-signaling for ODCs, we deleted insulin-like growth factor 1 receptor (IGF1R) specifically in mature ODCs of the mouse. We found that ODC survival and myelin status were unaffected by the absence of IGF1R until 15 months of age, indicating that IGF-1 signaling does not play a major role in post-mitotic ODCs during homeostasis. Notably, the absence of IGF1R did neither affect ODC survival nor myelin status upon cuprizone intoxication or induction of experimental autoimmune encephalomyelitis (EAE), models for toxic and autoimmune demyelination, respectively. Surprisingly, however, the absence of IGF1R from ODCs protected against clinical neuroinflammation in the EAE model. Together, our data indicate that IGF-1 signaling is not required for the function and survival of mature ODCs in steady-state and disease.

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.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Topography and evidence of a separate "fascia plate" for the femoral nerve inside the iliopsoas - A dorsal approach.

The femoral nerve stretch test is an essential part of clinical neurological examinations. This test is performed alongside Magnetic Resonance Imaging (MRI) to determine if there is any evidence of nerve root irritation, usually as a consequence of disc prolapse. The test occasionally gives false positive results. Why such false positives can occur, is subject to continued research, however, no obvious reason has yet emerged. We hypothesize that connectives of the femoral nerve may explain such a phenomenon. To see these connectives, we approached the femoral nerve from dorsal in 12 cases. With the use of ink injection into the subparaneural compartment of the femoral nerve and dissections, a thin transparent structure can clearly be seen that is separate from the epineurium, perineurium, and a paraneural sheath. A continuation of the paraneural sheath produces a fascia plate approximately 1.5 cm in width and with a thickness of around 3 mm, which not only circumnavigates the nerve but projects into the surrounding tissues. Our qualitative observations show that not only does this femoral nerve fascia plate exists, but it also contains nerves and vessels. Furthermore, we show that the femoral nerve is connected to the myofascial complex of the iliopsoas, and in a separate fascia plate from the iliopsoas fascia. This plate is a hitherto neglected connective which extends as far as the spinal dura mater. Evidence from our plastinates and histological sections suggests that when tension is applied to the femoral nerve during the femoral nerve stretch test, tension is also applied to the femoral nerve fascia plate. The femoral nerve fascia plate could be a specific factor that contributes to pain resulting in a false positive femoral nerve stretch test.

Kinetics of Equilibrium Passive Sampling of Organic Chemicals with Polymers in Diverse Mammalian Tissues.

Equilibrium passive sampling employing polydimethylsiloxane (PDMS) as a sampling phase can be used for the extraction of complex mixtures of organic chemicals from lipid-rich biota. We extended the method to lean tissues and more hydrophilic chemicals by implementing a mass-balance model for partitioning between lipids, proteins, and water in tissues and by accelerating uptake kinetics with a custom-built stirrer that effectively decreased time to equilibrium to less than 8 days even for a homogenized liver tissue with an only 4% lipid content. The partition constants log Klipid/PDMS between tissues and PDMS were derived from measured concentration in PDMS and the mass-balance model and were very similar for 40 neutral chemicals with octanol-water partition constants 1.4 < log Kow < 8.7, that is, log Klipid/PDMS of 1.26 (95% CI, 1.13-1.39) for the adipose tissue, 1.16 (1.00-1.33) for the liver, and 0.58 (0.42-0.73) for the brain. This conversion factor can be applied to interpret chemical analysis and in vitro bioassays after additionally accounting for a small fraction of coextracted lipids of <0.7% of the PDMS weight. PDMS is more widely applicable for passive sampling of mammalian tissues than previously thought, both, in terms of diversity of chemicals and the range of lipid contents of tissues and, therefore, an ideal method for human biomonitoring to be combined with in vitro bioassays.

Hypothalamic POMC neurons promote cannabinoid-induced feeding.

Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide ß-endorphin. CB1R activation selectively increases ß-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain.

Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation-if any-remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and ß-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and ß-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.

[Is there a new salivary gland? - Rather not!] / Gibt es eine neue Kopfspeicheldrüse? ­ Eher nicht!

In October 2020, the lay press, but also some medical journals and websites reported the putative discovery of a new salivary gland in the nasopharynx based on prostate-specific membrane antigen positron emission tomography computed tomography (PSMA-PET/CT) examinations. As an interdisciplinary group from the fields of anatomy, pathology, nuclear medicine and otorhinolaryngology, we come to the view that an accumulation of minor salivary glands has been described here. Minor salivary glands in the nasopharynx and in the peritubar region have been described at least since 1866. The current description in PSMA-PET/CT does not justify the definition of a new, independent salivary gland. The PSMA-PET/CT could, however, be suitable to better protect salivary glands in the nasopharynx when planning radiation therapy. This should be evaluated in clinical trials.

Dystrophic microglia in late-onset Alzheimer's disease.

Here, we summarize current understanding of functional involvement of microglial cells in the most common neurodegenerative disease to affect humans, which is sporadic or late-onset Alzheimer's disease (LOAD). Our review narrowly focuses on insights obtained from post-mortem neuropathological examinations of human brains paying particular attention to microglia as these cells have long been implicated as pivotal players in the cellular processes that lead to AD-type neurodegeneration. Although complete understanding of the roles played by microglia in AD neurodegeneration remains elusive, our studies thus far have illuminated microglial involvement in LOAD, showing that microglial dystrophy, the morphological manifestation of senescence, can be integrated with other hallmark pathological features of AD, such as intraneuronal neurofibrillary degeneration (NFD) and extracellular deposits of amyloid-beta (Aß) protein. We have demonstrated an in situ correlation between microglial dystrophy and presence of NFD suggesting that neurodegeneration is secondary to aging-related microglial deterioration, a concept founded on the notion that proper neuronal function is dependent on presence of healthy microglia. Diseased or weakened glia are detrimental for neuronal well-being because their ability to provide neuronal support may be impaired. Our most recent work also links microglial dystrophy with Aß deposits by showing that there is a chronic, yet futile microglial reaction to insoluble amyloid deposits. This inability of microglia to remove aggregated amyloid (a foreign body) causes microglial exhaustion and thereby exacerbates already ongoing aging-dependent microglial deterioration. An eventual total loss of functional microglia in advanced LOAD promotes widespread NFD, dementia, and brain failure.

Undisturbed climbing fiber pruning in the cerebellar cortex of CX3 CR1-deficient mice.

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now, the role of microglia in pruning remains under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the PC layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3 CR1) signaling during postnatal development using genetic ablation of the CX3 CR1 receptor and an in-depth histological analysis of the cerebellar cortex. We found an aberrant migration of microglia into the granule and the molecular layer. By electrophysiological analysis, we show that defective fractalkine signaling and the associated migration deficits neither affect the pruning of excess CFs nor the development of functional parallel fiber and inhibitory synapses with PCs. These findings indicate that CX3 CR1 signaling is not mandatory for correct cerebellar circuit formation. MAIN POINTS: Ablation of CX3 CR1 results in a transient migration defect in cerebellar microglia. CX3 CR1 is not required for functional pruning of cerebellar climbing fibers. Functional inhibitory and parallel fiber synapse development with Purkinje cells is undisturbed in CX3 CR1-deficient mice.

Fast microglial activation after severe traumatic brain injuries.

Traumatic brain injury is among the leading causes of death in individuals under 45 years of age. However, since trauma mechanisms and survival times differ enormously, the exact mechanisms leading to the primary and secondary injury and eventually to death after traumatic brain injury (TBI) remain unclear. Several studies showed the versatile functions of microglia, the innate macrophages of the brain, following a TBI. Earlier being characterized as rather neurotoxic, neuroprotective capacities were recently demonstrated, therefore, making microglia one of the key players following TBI. Especially in cases with only short survival times, immediate microglial reactions are of great forensic interest in questions of wound age estimation. Using standardized immunohistochemical methods, we examined 8 cases which died causatively of TBI with survival times between minutes and 7 days and 5 control cases with cardiovascular failure as the cause of death to determine acute changes in microglial morphology and antigen expression after TBI. In this pilot study, we detected highly localized changes in microglial morphology already early after traumatic damage, e.g., activated microglia and phagocyted erythrocytes in the contusion areas in cases with minute survival. Furthermore, an altered antigen expression was observed with increasing trauma wound age, showing similar effects like earlier transcriptomic studies. There is minute data on the direct impact of shear forces on microglial morphology. We were able to show localization-depending effects on microglial morphology causing localized dystrophy and adjacent activation. While rodent studies are widespread, they fail to mimic the exact mechanisms in human TBI response. Therefore, more studies focusing on cadaveric samples need to follow to thoroughly define the mechanisms leading to cell destruction and eventually evaluate their forensic value.

Tissue pretreatment for LC-MS/MS analysis of PUFA and eicosanoid distribution in mouse brain and liver.

Polyunsaturated fatty acids (PUFAs) and eicosanoids are important mediators of inflammation. The functional role of eicosanoids in metabolic-syndrome-related diseases has been extensively studied. However, their role in neuroinflammation and the development of neurodegenerative diseases is still unclear. The aim of this study was the development of a sample pretreatment protocol for the simultaneous analysis of PUFAs and eicosanoids in mouse liver and brain. Liver and brain samples of male wild-type C57BL/6J mice (11-122 mg) were used to investigate conditions for tissue rinsing, homogenization, extraction, and storage. A targeted liquid chromatography-negative electrospray ionization tandem mass spectrometry method was applied to quantify 7 PUFAs and 94 eicosanoids. The final pretreatment protocol consisted of a 5-min homogenization step by sonication in 650 µL n-hexane/2-propanol (60:40 v/v) containing 2,6-di-tert-butyl-4-methylphenol at 50 µg/mL. Homogenates representing 1 mg tissue were extracted in a single step with n-hexane/2-propanol (60:40 v/v) containing 0.1% formic acid. Autoxidation was prevented by addition of 2,6-di-tert-butyl-4-methylphenol at 50 µg/mL and keeping the samples at 4 °C during sample preparation. Extracts were dried under nitrogen and reconstituted in liquid chromatography eluent before analysis. Recovery was determined to range from 45% to 149% for both liver and brain tissue. Within-run and between-run variability ranged between 7% and 18% for PUFAs and between 1% and 24% for eicosanoids. In liver, 7 PUFAs and 15 eicosanoids were quantified; in brain, 6 PUFAs and 21 eicosanoids had significant differences within the brain substructures. In conclusion, a robust and reproducible sample preparation protocol for the multiplexed analysis of PUFAs and eicosanoids by liquid chromatography-tandem mass spectrometry in liver and discrete brain substructures was developed.

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.