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Background Cancer cachexia is a metabolic disorder characterized by the progressive loss of fat and lean mass that results in significant wasting, ultimately leading to reduced quality of life and increased mortality. Effective therapies for cachexia are lacking, potentially owing to the mismatch in clinically relevant models of cachexia. Specifically, cachexia observed in a clinical setting is commonly associated with advanced or late‐stage cancers that are metastatic, yet pre‐clinical metastatic models of cachexia are limited. Furthermore, the prevalence of cachexia in head and neck cancer patients is high, yet few pre‐clinical models of head and neck cancer cachexia exist. In addition to these shortcomings, cachexia is also heterogeneous among any given cancer, whereas patients with similar disease burden may experience significantly different degrees of cachexia symptoms. In order to address these issues, we characterize a metastatic model of human papilloma virus (HPV) positive head and neck squamous cell carcinoma that recapitulates the cardinal clinical and molecular features of cancer cachexia. Methods Male and female C57BL/6 mice were implanted subcutaneously with oropharyngeal squamous cell carcinoma cells stably transformed with HPV16 E6 and E7 together with hRas and luciferase (mEERL) that metastasizes to the lungs (MLM). We then robustly characterize the physiologic, behavioural, and molecular signatures during tumour development in two MLM subclones. Results Mice injected with MLM tumour cells rapidly developed primary tumours and eventual metastatic lesions to the lungs. MLM3, but not MLM5, engrafted mice progressively lost fat and lean mass during tumour development despite the absence of anorexia (P

Lipocalin 2 (LCN2) is a pleiotropic molecule that is induced in the central nervous system (CNS) in several acute and chronic pathologies. The acute induction of LCN2 evolved as a beneficial process, aimed at combating bacterial infection through the sequestration of iron from pathogens, while the role of LCN2 during chronic, non-infectious disease remains unclear, and recent studies suggest that LCN2 is neurotoxic. However, whether LCN2 is sufficient to induce behavioral and cognitive alterations remains unclear. In this paper, we sought to address the role of cerebral LCN2 on cognition in both acute and chronic settings. We demonstrate that LCN2 is robustly induced in the CNS during both acute and chronic inflammatory conditions, including LPS-based sepsis and cancer cachexia. In vivo, LPS challenge results in a global induction of LCN2 in the central nervous system, while cancer cachexia results in a distribution specific to the vasculature. Similar to these in vivo observations, in vitro modeling demonstrated that both glia and cerebral endothelium produce and secrete LCN2 when challenged with LPS, while only cerebral endothelium secrete LCN2 when challenged with cancer-conditioned medium. Chronic, but not short-term, cerebral LCN2 exposure resulted in reduced hippocampal neuron staining intensity, an increase in newborn neurons, microglial activation, and increased CNS immune cell infiltration, while gene set analyses suggested these effects were mediated through melanocortin-4 receptor independent mechanisms. RNA sequencing analyses of primary hippocampal neurons revealed a distinct transcriptome associated with prolonged LCN2 exposure, and ontology analysis was suggestive of altered neurite growth and abnormal spatial learning. Indeed, LCN2-treated hippocampal neurons display blunted neurite processes, and mice exposed to prolonged cerebral LCN2 levels experienced a reduction in spatial reference memory as indicated by Y-maze assessment. These findings implicate LCN2 as a pathologic mediator of cognitive decline in the setting of chronic disease.

Background Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. Methods TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. Results In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. Conclusion TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7’s neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7’s primary role in neuronal tissues is not related to antiviral immunity.

Microglia in the mediobasal hypothalamus (MBH) respond to inflammatory stimuli and metabolic perturbations to mediate body composition. This concept is well studied in the context of high fat diet induced obesity (HFDO), yet has not been investigated in the context of cachexia, a devastating metabolic syndrome characterized by anorexia, fatigue, and muscle catabolism. We show that microglia accumulate specifically in the MBH early in pancreatic ductal adenocarcinoma (PDAC)‐associated cachexia and assume an activated morphology. Furthermore, we observe astrogliosis in the MBH and hippocampus concurrent with cachexia initiation. We next show that circulating immune cells resembling macrophages infiltrate the MBH. PDAC‐derived factors induced microglia to express a transcriptional profile in vitro that was distinct from that induced by lipopolysaccharide (LPS). Microglia depletion through CSF1‐R antagonism resulted in accelerated cachexia onset and increased anorexia, fatigue, and muscle catabolism during PDAC. This corresponded with increased hypothalamic–pituitary–adrenal (HPA) axis activation. CSF1‐R antagonism had little effect on inflammatory response in the circulation, liver, or tumor. These findings demonstrate that microglia are protective against PDAC cachexia and provide mechanistic insight into this function., • Microgliosis occurs in the mediobasal hypothalamus early in pancreatic cancer.• Microglia respond to pancreatic cancer‐derived factors and produce arginase‐1.• Microglia depletion worsens cachexia during pancreatic cancer.

Voluntary wheel running is a valuable metabolic intervention and well-established measure of physical activity in preclinical rodent models. Herein, we describe detailed assembly instructions and provide necessary resources for researchers to build their own running wheels from commercial-off-the-shelf parts and an open-source program at approximately a tenth of the cost of commercially-available options.

Background: Cachexia-anorexia syndrome is a complex metabolic condition characterized by skeletal muscle wasting, reduced food intake and prominent involvement of systemic and central inflammation. Here, the gut barrier function was investigated in pancreatic cancer-induced cachexia mouse models by relating intestinal permeability to the degree of cachexia. We further investigated the involvement of the gut–brain axis and the crosstalk between tumour, gut and hypothalamus in vitro. Methods: Two distinct mouse models of pancreatic cancer cachexia (KPC and 4662) were used. Intestinal inflammation and permeability were assessed through fluorescein isothiocyanate dextran (FITC-dextran) and lipopolysaccharide (LPS), and hypothalamic and systemic inflammation through mRNA expression and plasma cytokines, respectively. To simulate the tumour–gut–brain crosstalk, hypothalamic (HypoE-N46) cells were incubated with cachexia-inducing tumour secretomes and LPS. A synthetic mimic of C26 secretome was produced based on its secreted inflammatory mediators. Each component of the mimic was systematically omitted to narrow down the key mediator(s) with an amplifying inflammation. To substantiate its contribution, cyclooxygenase-2 (COX-2) inhibitor was used. Results: In vivo experiments showed FITC-dextran was enhanced in the KPC group (362.3 vs. sham 111.4 ng/mL, P < 0.001). LPS was increased to 140.9 ng/mL in the KPC group, compared with sham and 4662 groups (115.8 and 115.8 ng/mL, P < 0.05). Hypothalamic inflammatory gene expression of Ccl2 was up-regulated in the KPC group (6.3 vs. sham 1, P < 0.0001, 4662 1.3, P < 0.001), which significantly correlated with LPS concentration (r = 0.4948, P = 0.0226). These data suggest that intestinal permeability is positively related to the cachexic degree. Prostaglandin E2 (PGE2) was confirmed to be present in the plasma and PGE2 concentration (log10) in the KPC group was much higher than in 4662 group (1.85 and 0.56 ng/mL, P < 0.001), indicating a role for PGE2 in pancreatic cancer-induced cachexia. Parallel to in vivo findings, in vitro experiments revealed that the cachexia-inducing tumour secretomes (C26, LLC, KPC and 4662) amplified LPS-induced hypothalamic IL-6 secretion (419%, 321%, 294%, 160%). COX-2 inhibitor to the tumour cells reduced PGE2 content (from 105 to 102 pg/mL) in the secretomes and eliminated the amplified hypothalamic IL-6 production. Moreover, results could be reproduced by addition of PGE2 alone, indicating that the increased hypothalamic inflammation is directly related to the PGE2 from tumour. Conclusions: PGE2 secreted by the tumour may play a role in amplifying the effects of bacteria-derived LPS on the inflammatory hypothalamic response. The cachexia-inducing potential of tumour mice models parallels the loss of intestinal barrier function. Tumour-derived PGE2 might play a key role in cancer-related cachexia-anorexia syndrome via tumour–gut–brain crosstalk.

Toll-like receptors 7 and 8 (TLR7 and TLR8) are endosomal pattern recognition receptors that detect a variety of single-stranded RNA species. While TLR7/8 agonists have robust therapeutic potential, clinical utility of these agents is limited by sickness responses associated with treatment induction. To understand the kinetics and mechanism of these responses, we characterized the acute and chronic effects of TLR7 stimulation. Single-cell RNA-sequencing studies, RNAscope, and radiolabeled in situ hybridization demonstrate that central nervous system gene expression of TLR7 is exclusive to microglia. In vitro studies demonstrate that microglia are highly sensitive to TLR7 stimulation, and respond in a dose-dependent manner to the imidazoquinoline R848. In vivo, both intraperitoneal (IP) and intracerebroventricular (ICV) R848 induce acute sickness responses including hypophagia, weight loss, and decreased voluntary locomotor activity, associated with increased CNS pro-inflammatory gene expression and changes to glial morphology. However, chronic daily IP R848 resulted in rapid tachyphylaxis of behavioral and molecular manifestations of illness. In microglial in vitro assays, pro-inflammatory transcriptional responses rapidly diminished in the context of repeated R848. In addition to TLR7 desensitization, we found that microglia become partially refractory to lipopolysaccharide (LPS) following R848 pretreatment, associated with induction of negative regulators A20 and Irak3. Similarly, mice pre-treated with R848 demonstrate reduced sickness responses, hypothalamic inflammation, and hepatic inflammation in response to LPS. These data combined demonstrate that TLR7 stimulation induces acute behavioral and molecular evidence of sickness responses. Following prolonged dosing, R848 induces a refractory state to both TLR7 and TLR4 activation, consistent with induced immune tolerance.

When energy balance is altered by aerobic exercise, starvation, and cold exposure, for example, there appears to be coordination of the responses of skeletal muscle, white adipose (WAT), and brown adipose (BAT) tissues. We hypothesized that WAT, BAT, and skeletal muscle may share an integrated regulation by the central nervous system (CNS); specifically, that neurons in brain regions associated with energy balance would possess neuroanatomical connections to permit coordination of multiple, complementary responses in these downstream tissues. To study this, we used trans-neuronal viral retrograde tract tracing, using isogenic strains of pseudorabies virus (PRV) with distinct fluorescent reporters (either eGFP or mRFP), injected pairwise into male rat gastrocnemius, subcutaneous WAT and interscapular BAT, coupled with neurochemical characterization of specific cell populations for cocaine- and amphetamine-related transcript (CART), oxytocin (OX), corticotrophin releasing hormone (CRH) and calcitonin gene-related peptide (CGRP). Cells in the paraventricular (PVN) and parabrachial (PBN) nuclei and brainstem showed dual projections to muscle + WAT, muscle + BAT, and WAT + BAT. Dual PRV-labeled cells were found in parvocellular, magnocellular and descending/pre-autonomic regions of the PVN, and multiple structural divisions of the PBN and brainstem. In most PBN subdivisions, more than 50% of CGRP cells dually projected to muscle + WAT and muscle + BAT. Similarly, 31–68% of CGRP cells projected both to WAT + BAT. However, dual PRV-labeled cells in PVN only occasionally expressed OX or CRH but not CART. These studies reveal for the first time both separate and shared outflow circuitries among skeletal muscle and subcutaneous WAT and BAT.

Control mice housed in the same room as mice with pancreatic ductal adenocarcinoma (PDAC) demonstrate decreased food intake coincident with the cachexia experienced by the mice with PDAC. Mice are considered an empathetic species, and we hypothesized that the reduced food intake in normal mice was an "empathy state" that was mediated by olfactory cues. Naïve male and female C57BL/6 mice were exposed to soiled bedding from mice experiencing PDAC induced cachexia or from control mice in the PDAC study. Body weight, food intake, and food spillage were measured across 48 h. Statistically significant differences in food consumption were found at various time points in both positive and negative directions for the 2 bedding conditions, and the direction of effect was opposite for males and females. Although analysis of data from previous PDAC studies showed differences in food spillage between PDAC mice and their controls, in this study we found no correlation between food consumption and food spillage based on bedding type. Disruption of food intake due to the "empathy state" requires testing larger numbers of animals to attain appropriate statistical power, which is contrary to the goal of using fewer animals. Empathy effects require careful consideration of sample size and cautious interpretation of results. This study also highlights the importance of sex as a biologic variable and why quantifying food spillage is important in studies of food intake.

Whether leptin acts in the paraventricular nucleus (PVN) to increase sympathetic nerve activity (SNA) is unclear, since PVN leptin receptors (LepR) are sparse. We show in rats that PVN leptin slowly increases SNA to muscle and brown adipose tissue, because it induces the expression of its own receptor and synergizes with local glutamatergic neurons. PVN LepR are not expressed in astroglia and rarely in microglia; instead, glutamatergic neurons express LepR, some of which project to a key presympathetic hub, the rostral ventrolateral medulla (RVLM). In PVN slices from mice expressing GCaMP6, leptin excites glutamatergic neurons. LepR are expressed mainly in thyrotropin-releasing hormone (TRH) neurons, some of which project to the RVLM. Injections of TRH into the RVLM and dorsomedial hypothalamus increase SNA, highlighting these nuclei as likely targets. We suggest that this neuropathway becomes important in obesity, in which elevated leptin maintains the hypothalamic pituitary thyroid axis, despite leptin resistance.

Weight loss and anorexia are common symptoms in cancer patients that occur prior to initiation of cancer therapy. Inflammation in the brain is a driver of these symptoms, yet cellular sources of neuroinflammation during malignancy are unknown. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), we observed early and robust myeloid cell infiltration into the brain. Infiltrating immune cells were predominately neutrophils, which accumulated at a unique central nervous system entry portal called the velum interpositum, where they expressed CCR2. Pharmacologic CCR2 blockade and genetic deletion of Ccr2 both resulted in significantly decreased brain-infiltrating myeloid cells as well as attenuated cachexia during PDAC. Lastly, intracerebroventricular blockade of the purinergic receptor P2RX7 during PDAC abolished immune cell recruitment to the brain and attenuated anorexia. Our data demonstrate a novel function for the CCR2/CCL2 axis in recruiting neutrophils to the brain, which drives anorexia and muscle catabolism., eLife digest Weight loss, decreased appetite and fatigue are symptoms of a wasting disorder known as cachexia, which is common in several serious diseases such as AIDS, chronic lung disease and heart failure. Up to 80 percent of people with advanced cancer also develop cachexia, and there are no effective treatments. It is not known how cachexia develops, but symptoms like appetite loss and fatigue are controlled by the brain. One theory is that the brain may be responding to a malfunctioning immune response that causes inflammation. While the brain was thought to be protected from this, new research has shown that it is possible for cells from the immune system to reach the brain in some conditions. To find out if this also happens in cancer, Burfeind et al. studied mice that had been implanted with pancreatic cancer cells and were showing signs of cachexia. Samples from the mice’s brains showed that immune cells known as neutrophils were present and active. A protein known as CCR2 was found in higher levels in the brains of these mice. This protein is involved in the movement of neutrophil cells through the body. To see what effect this protein had, Burfeind et al. gave the mice a drug that blocks CCR2. This prevented the neutrophils from entering the brain and reduced the symptoms of cachexia in the mice. To further confirm the role of CCR2, the mice were genetically modified so that they could not produce the protein. This reduced the number of neutrophils seen in the brain but not in the rest of the body. This suggests that a drug targeting CCR2 could help to reduce the symptoms of cachexia, without disrupting the normal immune response away from the brain. This approach would still need to be tested in clinical trials before it is possible to know how effective it might be in humans.

Background Up to 80% of pancreatic cancer patients suffer from cachexia, a devastating condition that exacerbates underlying disease, reduces quality of life, and increases treatment complications and mortality. Tumour‐induced inflammation is linked to this multifactorial wasting syndrome, but mechanisms and effective treatments remain elusive. Myeloid differentiation factor (MyD88), a key component of the innate immune system, plays a pivotal role in directing the inflammatory response to various insults. In this study, we tested whether MyD88 signalling is essential in the development of pancreatic cancer cachexia using a robust mouse tumour model. Methods Sex, age, and body weight‐matched wide type (WT) and MyD88 knockout (MyD88 KO) mice were orthotopically or intraperitoneally implanted with a pancreatic tumour cell line from a syngeneic C57BL/6 KRASG12D/+ P53R172H/+ Pdx‐Cre (KPC) mouse. We observed the effects of MyD88 signalling during pancreatic ductal adenocarcinoma progression and the cachexia development through behavioural, histological, molecular, and survival aspects. Results Blocking MyD88 signalling greatly ameliorated pancreatic ductal adenocarcinoma‐associated anorexia and fatigue, attenuated lean mass loss, reduced muscle catabolism and atrophy, diminished systemic and central nervous system inflammation, and ultimately improved survival. Our data demonstrate that MyD88 signalling plays a critical role in mediating pancreatic cancer‐induced inflammation that triggers cachexia development and therefore represents a promising therapeutic target. Conclusions MyD88‐dependent inflammation is crucial in the pathophysiology of pancreatic cancer progression and contributes to high mortality. Our findings implicate the importance of innate immune signalling pathways in pancreatic cancer cachexia and a novel therapeutic target.

Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.

Background Our evolutionary history is defined, in part, by our ability to survive times of nutrient scarcity. The outcomes of the metabolic and behavioural adaptations during starvation are highly efficient macronutrient allocation, minimization of energy expenditure, and maximized odds of finding food. However, in different contexts, caloric deprivation is met with vastly different physiologic and behavioural responses, which challenge the primacy of energy homeostasis. Methods We conducted a literature review of scientific studies in humans, laboratory animals, and non‐laboratory animals that evaluated the physiologic, metabolic, and behavioural responses to fasting, starvation, protein‐deficient or essential amino acid‐deficient diets, and cachexia. Studies that investigated the changes in ingestive behaviour, locomotor activity, resting metabolic rate, and tissue catabolism were selected as the focus of discussion. Results Whereas starvation responses prioritize energy balance, both protein malnutrition and cachexia present existential threats that induce unique adaptive programmes, which can exacerbate the caloric insufficiency of undernutrition. We compare and contrast the behavioural and metabolic responses and elucidate the mechanistic pathways that drive state‐dependent alterations in energy seeking and partitioning. Conclusions The evolution of energetically inefficient metabolic and behavioural responses to protein malnutrition and cachexia reveal a hierarchy of metabolic priorities governed by discrete regulatory networks.

Muscle atrophy occurs during chronic diseases, resulting in diminished quality of life and compromised treatment outcomes. There is a high demand for therapeutics that increase muscle mass while abrogating the need for special dietary and exercise requirements. Therefore, we developed an efficient nanomedicine approach capable of increasing muscle mass. Methods: The therapy is based on nanoparticle-mediated delivery of follistatin messenger RNA (mRNA) to the liver after subcutaneous administration. The delivered mRNA directs hepatic cellular machinery to produce follistatin, a glycoprotein that increases lean mass through inhibition of negative regulators of muscle mass (myostatin and activin A). These factors are elevated in numerous disease states, thereby providing a target for therapeutic intervention. Results: Animal studies validated that mRNA-loaded nanoparticles enter systemic circulation following subcutaneous injection, accumulate and internalize in the liver, where the mRNA is translated into follistatin. Follistatin serum levels were elevated for 72 h post injection and efficiently reduced activin A and myostatin serum concentrations. After eight weeks of repeated injections, the lean mass of mice in the treatment group was ~10% higher when compared to that of the controls. Conclusion: Based on the obtained results demonstrating an increased muscle mass as well as restricted fat accumulation, this nanoplatform might be a milestone in the development of mRNA technologies and the treatment of muscle wasting disorders.

A priority in cancer research is to innovate therapies that are not only effective against tumor progression but also address comorbidities such as cachexia that limit quality and quantity of life. We demonstrate that TLR7/8 agonist R848 induces anti-tumor responses and attenuates cachexia in murine models of pancreatic ductal adenocarcinoma (PDAC). In vivo, tumors from two of three cell lines were R848-sensitive, resulting in smaller tumor mass, increased immune complexity, increased CD8

Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC.

Purpose We determine if monomethyl fumarate (MMF) can protect the retina in mice subjected to light-induced retinopathy (LIR). Methods Albino BALB/c mice were intraperitoneally injected with 50 to 100 mg/kg MMF before or after exposure to bright white light (10,000 lux) for 1 hour. Seven days after light exposure, retinal structure and function were evaluated by optical coherence tomography (OCT) and electroretinography (ERG), respectively. Retinal histology also was performed to evaluate photoreceptor loss. Expression levels of Hcar2 and markers of microglia activation were measured by quantitative PCR (qPCR) in the neural retina with and without microglia depletion. At 24 hours after light exposure, retinal sections and whole mount retinas were stained with Iba1 to evaluate microglia status. The effect of MMF on the nuclear factor kB subunit 1 (NF-kB) and Nrf2 pathways was measured by qPCR and Western blot. Results MMF administered before light exposure mediated dose-dependent neuroprotection in a mouse model of LIR. A single dose of 100 mg/kg MMF fully protected retinal structure and function without side effects. Expression of the Hcar2 receptor and the microglia marker Cd14 were upregulated by LIR, but suppressed by MMF. Depleting microglia reduced Hcar2 expression and its upregulation by LIR. Microglial activation, upregulation of proinflammatory genes (Nlrp3, Caspase1, Il-1β, Tnf-α), and upregulation of antioxidative stress genes (Hmox1) associated with LIR were mitigated by MMF treatment. Conclusions MMF can completely protect the retina from LIR in BALB/c mice. Expression of Hcar2, the receptor of MMF, is microglia-dependent in the neural retina. MMF-mediated neuroprotection was associated with attenuation of microglia activation, inflammation and oxidative stress in the retina.

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance-like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation.

Objective Activation of hypothalamic agouti-related peptide (AgRP)+ve neurons during energy deficit is a negative valence signal, rapidly activating food seeking behaviors. This study examined the roles of MC3Rs expressed by AgRP+ve neurons. Methods AgRP-MC3R mice expressing MC3Rs selectively in AgRP+ve neurons were generated by crossing AgRP-IRES-Cre mice with LoxTBMc3r mice containing a “loxP-STOP-loxP” sequence in the 5’ UTR. Body weight, body composition and feeding behavior were assessed during ad libitum and timed-restricted feeding conditions. Results In females, food intake of AgRP-IRES-Cre(+ve) (n=7) or AgRP-IRES-Cre(-ve) (n=9) mice was not significantly different; these mice were therefore pooled to form the “control” group. Female AgRP-MC3R mice exhibited lower food intake (25.4±2.4 kJ/12h, n=6) compared to controls (35.3±1.8 kJ/12h, n=16) and LoxTBMc3r mice (32.1±2.1 kJ/12h, n=9) in the active phase during the dark period. Food intake during the rest phase (lights-on) when mice consume less food (9–10 kJ) was normal between genotypes. Body weight and composition of AgRP-MC3R and LoxTBMc3r mice was similar, suggesting compensatory mechanisms for reduced calorie intake. Remarkably, AgRP-MC3R mice continued to consume less food during re-feeding after fasting and timed-restricted feeding. Conclusions MC3Rs expressed on AgRP+ve neurons appear to exert a strong inhibitory signal on hypothalamic networks governing feeding behavior.

Hypothalamic inflammation is a key component of acute sickness behavior and cachexia, yet mechanisms of inflammatory signaling in the central nervous system remain unclear. Previous work from our lab and others showed that while MyD88 is an important inflammatory signaling pathway for sickness behavior, MyD88 knockout (MyD88KO) mice still experience sickness behavior after inflammatory stimuli challenge. We found that after systemic lipopolysaccharide (LPS) challenge, MyD88KO mice showed elevated expression of several cytokine and chemokine genes in the hypothalamus. We therefore assessed the role of an additional inflammatory signaling pathway, TRIF, in acute inflammation (LPS challenge) and in a chronic inflammatory state (cancer cachexia). TRIFKO mice resisted anorexia and weight loss after peripheral (intraperitoneal, IP) or central (intracerebroventricular, ICV) LPS challenge and in a model of pancreatic cancer cachexia. Compared to WT mice, TRIFKO mice showed attenuated upregulation of Il6, Ccl2, Ccl5, Cxcl1, Cxcl2, and Cxcl10 in the hypothalamus after IP LPS treatment, as well as attenuated microglial activation and neutrophil infiltration into the brain after ICV LPS treatment. Lastly, we found that TRIF was required for Ccl2 upregulation in the hypothalamus and induction of the catabolic genes, Mafbx, Murf1, and Foxo1 in gastrocnemius during pancreatic cancer. In summary, our results show that TRIF is an important inflammatory signaling mediator of sickness behavior and cachexia and presents a novel therapeutic target for these conditions.

Peak bone mass, one of the most important predictors for fracture risk later in life, is attained during puberty and adolescence and influenced by neonatal and pubertal sex-specific gonadal hormones and GH-IGF-I secretion patterns. This study examined the effects of brief neonatal estrogen (NE) exposure on growth and skeletal development in C57BL/6J mice. A single injection of 100-μg estradiol or vehicle was administered on the first day of life. Growth parameters were monitored and skeletal phenotyping performed at 16 weeks in female mice and at 4 and 16 weeks in male mice. NE exposure negatively impacted adult femoral length in both sexes, but adult body weight, areal bone density, and bone strength in female mice were unaffected. In contrast, somatic growth was attenuated in estrogen-exposed male mice throughout the study period. At the prepubertal time point, the estrogen-exposed males exhibited higher bone mineral density, cortical volume, and cortical thickness compared with controls. However, by the time of peak bone mass acquisition, the early skeletal findings had reversed; estrogen-exposed mice had lower bone density with reduced cross-sectional area, cortical volume, and cortical thickness, resulting in cortical bones that were less resistant to fracture. NE exposure also resulted in reduced testicular volume and lower circulating IGF-I. Male mice exposed to estrogen on the first day of life experience age-dependent changes in skeletal development. Prepubertal animals experience greater endocortical bone acquisition as a result of estrogen exposure. However, by adulthood, continued developmental changes result in overall reduced skeletal integrity.

Background During acute infections and chronic illnesses, the pro-inflammatory cytokine interleukin-1β (IL-1β) acts within the brain to elicit metabolic derangements and sickness behaviors. It is unknown which cells in the brain are the proximal targets for IL-1β with respect to the generation of these illness responses. We performed a series of in vitro experiments to (1) investigate which brain cell populations exhibit inflammatory responses to IL-1β and (2) examine the interactions between different IL-1β-responsive cell types in various co-culture combinations. Methods We treated primary cultures of murine brain microvessel endothelial cells (BMEC), astrocytes, and microglia with PBS or IL-1β, and then performed qPCR to measure inflammatory gene expression or immunocytochemistry to evaluate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. To evaluate whether astrocytes and/or BMEC propagate inflammatory signals to microglia, we exposed microglia to astrocyte-conditioned media and co-cultured endothelial cells and glia in transwells. Treatment groups were compared by Student’s t tests or by ANOVA followed by Bonferroni-corrected t tests. Results IL-1β increased inflammatory gene expression and NF-κB activation in primary murine-mixed glia, enriched astrocyte, and BMEC cultures. Although IL-1β elicited minimal changes in inflammatory gene expression and did not induce the nuclear translocation of NF-κB in isolated microglia, these cells were more robustly activated by IL-1β when co-cultured with astrocytes and/or BMEC. We observed a polarized endothelial response to IL-1β, because the application of IL-1β to the abluminal endothelial surface produced a more complex microglial inflammatory response than that which occurred following luminal IL-1β exposure. Conclusions Inflammatory signals are detected, amplified, and propagated through the CNS via a sequential and reverberating signaling cascade involving communication between brain endothelial cells and glia. We propose that the brain’s innate immune response differs depending upon which side of the blood-brain barrier the inflammatory stimulus arises, thus allowing the brain to respond differently to central vs. peripheral inflammatory insults. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0908-4) contains supplementary material, which is available to authorized users.

Background The physiological and behavioral symptoms of sickness, including fever, anorexia, behavioral depression, and weight loss can be both beneficial and detrimental. These sickness responses are triggered by pro-inflammatory cytokines acting on cells within the brain. Previous research demonstrates that the febrile response to peripheral insults depends upon prostaglandin production by vascular endothelial cells, but the mechanisms and specific cell type(s) responsible for other sickness responses remain unknown. The purpose of the present study was to identify which cells within the brain are required for sickness responses triggered by central nervous system inflammation. Methods Intracerebroventricular (ICV) administration of 10 ng of the potent pro-inflammatory cytokine interleukin-1β (IL-1β) was used as an experimental model of central nervous system cytokine production. We examined which cells respond to IL-1β in vivo via fluorescent immunohistochemistry. Using multiple transgenic mouse lines expressing Cre recombinase under the control of cell-specific promoters, we eliminated IL-1β signaling from different populations of cells. Food consumption, body weight, movement, and temperature were recorded in adult male mice and analyzed by two-factor ANOVA to determine where IL-1β signaling is essential for sickness responses. Results Endothelial cells, microglia, ependymal cells, and astrocytes exhibit nuclear translocation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in response to IL-1β. Interfering with IL-1β signaling in microglia, endothelial cells within the parenchyma of the brain, or both did not affect sickness responses. Only mice that lacked IL-1β signaling in all endothelium including fenestrated capillaries lacked sickness responses. Conclusions These experiments show that IL-1β-induced sickness responses depend on intact IL-1β signaling in blood vessels and suggest that fenestrated capillaries act as a critical signaling relay between the immune and nervous systems. Trial registration Not applicable. Electronic supplementary material The online version of this article (10.1186/s12974-017-0990-7) contains supplementary material, which is available to authorized users.

Background Cachexia is a complex metabolic and behavioural syndrome lacking effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is one of the most important conditions associated with cachexia, with >80% of PDAC patients suffering from the condition. To establish the cardinal features of a murine model of PDAC‐associated cachexia, we characterized the effects of implanting a pancreatic tumour cell line from a syngeneic C57BL/6 KRASG12D P53R172H Pdx‐Cre+/+ (KPC) mouse. Methods Male and female C57BL/6 mice were inoculated subcutaneously, intraperitoneally, or orthotopically with KPC tumour cells. We performed rigorous phenotypic, metabolic, and behavioural analysis of animals over the course of tumour development. Results All routes of administration produced rapidly growing tumours histologically consistent with moderate to poorly differentiated PDAC. The phenotype of this model was dependent on route of administration, with orthotopic and intraperitoneal implantation inducing more severe cachexia than subcutaneous implantation. KPC tumour growth decreased food intake, decreased adiposity and lean body mass, and decreased locomotor activity. Muscle catabolism was observed in both skeletal and cardiac muscles, but the dominant catabolic pathway differed between these tissues. The wasting syndrome in this model was accompanied by hypothalamic inflammation, progressively decreasing brown and white adipose tissue uncoupling protein 1 (Ucp1) expression, and increased peripheral inflammation. Haematological and endocrine abnormalities included neutrophil‐dominant leukocytosis and anaemia, and decreased serum testosterone. Conclusions Syngeneic KPC allografts are a robust model for studying cachexia, which recapitulate key features of the PDAC disease process and induce a wide array of cachexia manifestations. This model is therefore ideally suited for future studies exploring the physiological systems involved in cachexia and for preclinical studies of novel therapies.

Fatigue is the most common symptom related to cytotoxic chemotherapeutic treatment of cancer. Peripheral inflammation associated with cytotoxic chemotherapy is likely a causal factor of fatigue. The neural mechanisms by which cytotoxic chemotherapy associated inflammation induces fatigue behavior are not known. This lack of knowledge hinders development of interventions to reduce or prevent this disabling symptom. Infection induced fatigue/lethargy in rodents is mediated by suppression of hypothalamic orexin activity. Orexin is critical for maintaining wakefulness and motivated behavior. Though there are differences between infection and cytotoxic chemotherapy in some symptoms, both induce peripheral inflammation and fatigue. Based on these similarities we hypothesized that cytotoxic chemotherapy induces fatigue by disrupting orexin neuron activity. We found that a single dose of a cytotoxic chemotherapy cocktail (cyclophosphamide, adriamycin, 5-fluorouracil—CAF) induced fatigue/lethargy in mice and rats as evidenced by a significant decline in voluntary locomotor activity measured by telemetry. CAF induced inflammatory gene expression—IL-1R1 (p

Hyperglycemia worsens stroke, yet rigorous glycemic control does not improve neurologic outcome. An alternative is to target downstream molecular mediator(s) triggered by hyperglycemia but independent of prevailing glycemia. Soluble epoxide hydrolase (sEH) is a potential mediator of injury via its metabolism of neuroprotective epoxyeicosatrienoic acids (EETs). We tested whether hyperglycemia exacerbates cerebral injury by upregulating sEH and decreasing brain EET levels. Type 1 diabetes mellitus was modeled by streptozotocin (STZ;50 mg/kg per day intraperitoneally, 5 days) in male mice. At 4 weeks, STZ-treated and control mice underwent 45-minute middle cerebral artery occlusion (MCAO) with or without sEH blockade by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB;1 mg/kg intraperitoneally daily for 6 days before MCAO). The STZ-treated mice had increased sEH mRNA expression in cerebral vessels and decreased EET concentrations in brain. There was no difference in cortical perfusion between groups. The STZ-treated mice sustained larger brain infarct than controls. Pretreatment with t-AUCB eliminated the difference in infarct size and EETs concentration between STZ-treated mice and controls, without altering glycemia. We conclude that type 1 diabetes mellitus upregulates sEH mRNA and decreases concentrations of neuroprotective EETs within the brain, leading to worse stroke outcome. The data indicate that sEH antagonism may be beneficial in the setting of hyperglycemic stroke.

Cachexia is a wasting condition defined by skeletal muscle atrophy in the setting of systemic inflammation. To explore the site at which inflammatory mediators act to produce atrophy in vivo, we utilized mice with a conditional deletion of the inflammatory adaptor protein myeloid differentiation factor 88 (MyD88). Although whole-body MyD88-knockout (wbMyD88KO) mice resist skeletal muscle atrophy in response to LPS, muscle-specific deletion of MyD88 is not protective. Furthermore, selective reexpression of MyD88 in the muscle of wbMyD88KO mice via electroporation fails to restore atrophy gene induction by LPS. To evaluate the role of glucocorticoids as the inflammation-induced mediator of atrophy in vivo, we generated mice with targeted deletion of the glucocorticoid receptor in muscle (mGRKO mice). Muscle-specific deletion of the glucocorticoid receptor affords a 71% protection against LPS-induced atrophy compared to control animals. Furthermore, mGRKO mice exhibit 77% less skeletal muscle atrophy than control animals in response to tumor growth. These data demonstrate that glucocorticoids are a major determinant of inflammation-induced atrophy in vivo and play a critical role in the pathogenesis of endotoxemic and cancer cachexia.—Braun, T. P., Grossberg, A. J., Krasnow, S. M., Levasseur, P. R., Szumowski, M., Zhu, X. X., Maxson, J. E., Knoll, J. G., Barnes, A. P., and Marks, D. L. Cancer- and endotoxin-induced cachexia require intact glucocorticoid signaling in skeletal muscle.

Melanocortin-3 receptors (MC3R) have a contextual role in appetite control that is amplified with hypocaloric conditioning. C57BL/6J (B6) mice subjected to hypocaloric feeding schedules (HFS) exhibit compulsive behavioral responses involving food anticipatory activity (FAA) and caloric loading following food access. These homeostatic responses to calorie-poor environs are attenuated in B6 mice in which Mc3r transcription is suppressed by a lox-stop-lox sequence in the 5’UTR (Mc3rTB/TB ). Here, we report that optimization of caloric loading in B6 mice subject to HFS, characterized by increased meal size and duration, is not observed in Mc3rTB/TB mice. Analysis of hypothalamic and neuroendocrine responses to HFS throughout the light-dark cycle suggests uncoupling of hypothalamic responses involving appetite-stimulating fasting-responsive hypothalamic neurons expressing agouti-related peptide (AgRP) and neuropeptide Y (Npy). Rescuing Mc3rs expression in Nkx2.1(+ve) neurons is sufficient to restore normal hypothalamic responses to negative energy balance. In addition, Mc3rs expressed in Nkx2.1(+ve) neurons are also sufficient to restore FAA and caloric loading of B6 mice subjected to HFS. In summary, MC3Rs expressed in Nkx2.1(+ve) neurons are sufficient to coordinate hypothalamic response and expression of compulsive behavioral responses involving meal anticipation and consumption of large meals during situations of prolonged negative energy balance.

Mesenchymal stromal cells (MSCs) present in the bone marrow microenvironment secrete cytokines and angiogenic factors that support the maintenance and regenerative expansion of hematopoietic stem and progenitor cells (HSPCs). Here, we tested the hypothesis that extracellular vesicles (EVs) released by MSCs contribute to the paracrine crosstalk that shapes hematopoietic function. We systematically characterized EV release by murine stromal cells and demonstrate that MSC-derived EVs prompt a loss of HSPC quiescence with concomitant expansion of murine myeloid progenitors. Our studies reveal that HSPC expansion by MSC EVs is mediated via the MyD88 adapter protein and is partially blocked by treatment with a TLR4 inhibitor. Imaging of fluorescence protein-tagged MSC EVs corroborated their cellular co-localization with TLR4 and endosomal Rab5 compartments in HSPCs. The dissection of downstream responses to TLR4 activation reveals that the mechanism by which MSC EVs impact HSPCs involves canonical NF-κB signaling and downstream activation of Hif-1α and CCL2 target genes. Our aggregate data identify a previously unknown role for MSC-derived EVs in the regulation of hematopoiesis through innate immune mechanisms and illustrate the expansive cell-cell crosstalk in the bone marrow microenvironment.

Signs and symptoms of multiple sclerosis are usually attributed to demyelinating lesions in the spinal cord or cerebral cortex. The hypothalamus is a region that is often overlooked yet controls many important homeostatic functions, including those that are perturbed in multiple sclerosis. In this review we discuss how hypothalamic dysfunction may contribute to signs and symptoms in people with multiple sclerosis. While dysfunction of the hypothalamic-pituitary-adrenal axis is common in multiple sclerosis, the effects and mechanisms of this dysfunction are not well understood. We discuss three hypothalamic mechanisms of fatigue in multiple sclerosis: (1) general hypothalamic-pituitary-adrenal axis hyperactivity, (2) disordered orexin neurotransmission, (3) abnormal cortisol secretion. We then review potential mechanisms of weight dysregulation caused by hypothalamic dysfunction. Lastly, we propose future studies and therapeutics to better understand and treat hypothalamic dysfunction in multiple sclerosis. Hypothalamic dysfunction appears to be common in multiple sclerosis, yet current studies are underpowered and contradictory. Future studies should contain larger sample sizes and standardize hormone and neuropeptide measurements.

Leukemia inhibitory factor (LIF) has been suggested to function as a potent inhibitor of feed intake in rodents. In sheep, intravenous injection of lipopolysaccharide (LPS) resulted in an increase in gene expression for LIF in the arcuate nucleus ( < 0.01). In the same experiment, agouti related protein (AgRP) expression was elevated ( < 0.05) but there were no effects on proopiomelanocortin expression. Another group of sheep were provided intracerebroventricular (ICV) injections of LIF at 250, 500, 1,000, and 2,500 ng per sheep. Cumulative feed intake was inhibited by the 1,000- and 2,500-ng doses at 8 and 10 h after ICV injection ( < 0.03). All doses of LIF elevated temperature above 40°C, indicating a fever. When AgRP was intracerebroventricularly injected before LIF, there was no effect of LIF to reduce feed intake, suggesting the LIF inhibition of feed intake is consistent with the concept that the effect is mediated by the melanocortin-4 receptor. In an experiment to determine whether endocrine and metabolic effects of LIF were similar to reported effects of LPS, sheep were intracerebroventricularly injected with 2,500 ng LIF, and blood samples were collected at 10-min intervals for 6 h for assay of LH, samples from the first 3 h were assayed for GH, and samples at 30-min intervals were assayed for glucose and free fatty acids. The effect of treatment and treatment × time interaction was significant, indicating elevated plasma free fatty acids ( < 0.03 and < 0.001, respectively) and glucose ( < 0.01 and < 0.0001, respectively). There was also a treatment × time interaction on circulating concentrations of LH such that LIF caused LH to decrease ( < 0.0001). Additionally, there was a tendency for LIF treatment to increase circulating concentrations of GH (P = 0.0874). The effects of LIF on feed intake and other parameters was similar to the effects of LPS and leads to a hypothesis that LIF expression in response to LPS may be a component of the mechanism for feed intake inhibition and perhaps for changes in selected hormone and metabolites in disease models.

Sickness behaviors and metabolic responses to invading pathogens are common to nearly all types of infection. These responses evolved to provide short-term benefit to the host to ward off infection, but impact on quality of life and when prolonged lead to neurodegeneration, depression and cachexia. Among the major infectious agents, viruses most frequently enter the brain, resulting in profound neuroinflammation. We sought to define the unique features of the inflammatory response in the brain to these infections. We demonstrate that the molecular pathway defining the central response to dsRNA is distinct from that found in the periphery. The behavioral and physical response to the dsRNA mimetic poly I:C is dependent on signaling via MyD88 when it is delivered centrally, whereas this response is mediated via the TRIF pathway when delivered peripherally. We also define the likely cellular candidates for this MyD88-dependent step. These findings suggest that symptom management is possible without ameliorating protective antiviral immune responses.

Animals exhibit a rapid and sustained anorexia when fed a diet that is deficient in a single indispensable amino acid (IAA). The chemosensor for IAA deficiency resides within the anterior piriform cortex (APC). Although the cellular and molecular mechanisms by which the APC detects IAA deficiency are well established, the efferent neural pathways that reduce feeding in response to an IAA-deficient diet remain to be fully characterized. In the present work, we investigated whether 1) central melanocortin signaling is involved in IAA deficiency-induced anorexia (IAADA) and 2) IAADA engages other key appetite-regulating neuronal populations in the hypothalamus. Rats and mice that consumed a valine-deficient diet (VDD) for 2–3 wk exhibited marked reductions in food intake, body weight, fat and lean body mass, body temperature, and white adipose tissue leptin gene expression, as well as a paradoxical increase in brown adipose tissue uncoupling protein-1 mRNA. Animals consuming the VDD had altered hypothalamic gene expression, typical of starvation. Pharmacological and genetic blockade of central melanocortin signaling failed to increase long-term food intake in this model. Chronic IAA deficiency was associated with a marked upregulation of corticotropin-releasing hormone expression in the lateral hypothalamus, particularly in the parasubthalamic nucleus, an area heavily innervated by efferent projections from the APC. Our observations indicate that the hypothalamic melanocortin system plays a minor role in acute, but not chronic, IAADA and suggest that the restraint on feeding is analogous to that observed after chronic dehydration.

Adipose tissue macrophages are important mediators of inflammation and insulin resistance in obesity. IFN-γ is a central regulator of macrophage function. The role of IFN-γ in regulating systemic inflammation and insulin resistance in obesity is unknown. We studied obese IFN-γ knockout mice to identify the role of IFN-γ in regulating inflammation and insulin sensitivity in obesity. IFN-γ-knockout C57Bl/6 mice and wild-type control litter mates were maintained on normal chow or a high fat diet for 13 weeks and then underwent insulin sensitivity testing then sacrifice and tissue collection. Flow cytometry, intracellular cytokine staining, and QRTPCR were used to define tissue lymphocyte phenotype and cytokine expression profiles. Adipocyte size was determined from whole adipose tissue explants examined under immunofluorescence microscopy. Diet-induced obesity induced systemic inflammation and insulin resistance, along with a pan-leukocyte adipose tissue infiltrate that includes macrophages, T-cells, and NK cells. Obese IFN-γ-knockout animals, compared with obese wild-type control animals, demonstrate modest improvements in insulin sensitivity, decreased adipocyte size, and an M2-shift in ATM phenotype and cytokine expression. These data suggest a role for IFN-γ in the regulation of inflammation and glucose homeostasis in obesity though multiple potential mechanisms, including effects on adipogenesis, cytokine expression, and macrophage phenotype.

Systemic and CNS-delimited inflammation triggers skeletal muscle catabolism in a manner dependent on glucocorticoid signaling., Skeletal muscle catabolism is a co-morbidity of many chronic diseases and is the result of systemic inflammation. Although direct inflammatory cytokine action on muscle promotes atrophy, nonmuscle sites of action for inflammatory mediators are less well described. We demonstrate that central nervous system (CNS)–delimited interleukin 1β (IL-1β) signaling alone can evoke a catabolic program in muscle, rapidly inducing atrophy. This effect is dependent on hypothalamic–pituitary–adrenal (HPA) axis activation, as CNS IL-1β–induced atrophy is abrogated by adrenalectomy. Furthermore, we identified a glucocorticoid-responsive gene expression pattern conserved in models of acute and chronic inflammatory muscle atrophy. In contrast with studies suggesting that the direct action of inflammatory cytokines on muscle is sufficient to induce catabolism, adrenalectomy also blocks the atrophy program in response to systemic inflammation, demonstrating that glucocorticoids are requisite for this process. Additionally, circulating levels of glucocorticoids equivalent to those produced under inflammatory conditions are sufficient to cause profound muscle wasting. Together, these data suggest that a significant component of inflammation-induced muscle catabolism occurs indirectly via a relay in the CNS.

The melanocortin-3 receptor (MC3R) gene is pleiotropic, influencing body composition, natriuresis, immune function, and entrainment of circadian rhythms to nutrient intake. MC3Rs are expressed in hypothalamic and limbic regions of the brain and in peripheral tissues. To investigate the roles of central MC3Rs, we inserted a "lox-stop-lox" (LoxTB) 5' of the translation initiation codon of the mouse Mc3r gene and reactivated transcription using neuron-specific Cre transgenic mice. As predicted based on earlier observations of Mc3r knock-out mice, Mc3r(TB/TB) mice displayed reduced lean mass, increased fat mass, and accelerated diet-induced obesity. Surprisingly, rescuing Mc3r expression in the nervous system using the Nestin-Cre transgene only partially rescued obesity in chow-fed conditions and had no impact on the accelerated diet-induced obesity phenotype. The ventromedial hypothalamus (VMH), a critical node in the neural networks regulating feeding-related behaviors and metabolic homeostasis, exhibits dense Mc3r expression relative to other brain regions. To target VMH MC3R expression, we used the steroidogenic factor-1 Cre transgenic mouse. Although restoring VMH MC3R signaling also had a modest impact on obesity, marked improvements in metabolic homeostasis were observed. VMH MC3R signaling was not sufficient to rescue the lean mass phenotype or the regulation of behaviors anticipating food anticipation. These results suggest that actions of MC3Rs impacting on energy homeostasis involve both central and peripheral sites of action. The impact of central MC3Rs on behavior and metabolism involves divergent pathways; VMH MC3R signaling improves metabolic homeostasis but does not significantly impact on the expression of behaviors anticipating nutrient availability.

Exposure to chronic and acute oxidative stress is correlated with many human diseases, including, but not limited to, cancer, heart disease, diabetes, and obesity. In addition to cellular lipids and proteins, cellular oxidative stress can result in damage to DNA bases, especially in mitochondrial DNA. We previously described the development of spontaneous late-onset obesity, hepatic steatosis, hyperinsulinemia, and hyperleptinemia in mice that are deficient in the DNA glycosylase nei-like 1 (NEIL1), which initiates base excision repair of several oxidatively damaged bases. In the current study, we report that exposure to a chronic oxidative stress in the form of a high-fat diet greatly accelerates the development of obesity in neil1−/−mice. Following a 5-wk high-fat diet challenge, neil1−/−mice gained significantly more body weight than neil1+/+littermates and had increased body fat accumulation and moderate to severe hepatic steatosis. Analysis of oxygen consumption by indirect calorimetry indicated a modest reduction in total oxygen consumption in neil1−/−mice that was abolished upon correction for lean body mass. Additionally, hepatic expression of several inflammatory genes was significantly upregulated in neil1−/−mice following high-fat diet challenge compared with chow-fed or neil1+/+counterparts. A long-term high-fat diet also induced glucose intolerance as well as a significant reduction in mitochondrial DNA and protein content in neil1−/−mice. Collectively, these data indicate that NEIL1 deficiency results in an increased susceptibility to obesity and related complications potentially by lowering the threshold for tolerance of cellular oxidative stress in neil1−/−mice.

Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.

Sensitive assays for rapid quantitative analysis of histologic sections, resected tissue specimens, or in situ tissue are highly desired for early disease diagnosis. Stained histopathology is the gold standard but remains a subjective practice on processed tissue taking from hours to days. We describe a microscopy technique that obtains a sensitive and accurate color-coded image from intrinsic molecular markers. Spectrally reconstructed nonlinear interferometric vibrational imaging can differentiate cancer versus normal tissue sections with greater than 99% confidence interval in a preclinical rat breast cancer model and define cancer boundaries to ±100 μm with greater than 99% confidence interval, using fresh unstained tissue sections imaged in less than 5 minutes. By optimizing optical sources and beam delivery, this technique can potentially enable real-time point-of-care optical molecular imaging and diagnosis. Cancer Res; 70(23); 9562–9. ©2010 AACR.

The central melanocortin system plays a key role in the regulation of food intake and energy homeostasis. We investigated whether genetic or pharmacologic blockade of central melanocortin signaling attenuates cardiac cachexia in mice and rats with heart failure. Permanent ligation of the left coronary artery (myocardial infarction (MI)) or sham operation was performed in wild-type (WT) or melanocortin-4 receptor (MC4R) knockout mice. Eight weeks after surgery, WT-Sham mice had significant increases in lean body mass (LBM; PPPPPPP

The hypothalamic melanocortin system, which controls appetite and energy expenditure, develops during the third trimester in primates. Thus, maternal nutrition and health may have a profound influence on the development of this system. To study the effects of chronic maternal high-fat diet (HFD) on the development of the melanocortin system in the fetal nonhuman primate, we placed adult female macaques on either a control (CTR) diet or a HFD for up to 4 yr. A subgroup of adult female HFD animals was also switched to CTR diet during the fifth year of the study (diet reversal). Third-trimester fetuses from mothers on HFD showed increases in proopiomelanocortin mRNA expression, whereas agouti-related protein mRNA and peptide levels were decreased in comparison with CTR fetuses. Proinflammatory cytokines, including IL-1β and IL-1 type 1 receptor, and markers of activated microglia were elevated in the hypothalamus, suggesting an activation of the local inflammatory response. Fetuses of diet-reversal mothers had normal melanocortin levels. These results raise the concern that chronic consumption of a HFD during pregnancy, independent of maternal obesity and diabetes, can lead to widespread activation of proinflammatory cytokines that may alter the development of the melanocortin system. The abnormalities in the fetal POMC system, if maintained into the postnatal period, could impact several systems, including body weight homeostasis, stress responses, and cardiovascular function. Indeed, the HFD offspring develop early-onset excess weight gain. These abnormalities may be prevented by healthful nutrient consumption during pregnancy even in obese and severely insulin-resistant individuals.

Inflammation-associated cachexia is associated with multiple chronic diseases and involves activation of appetite regulating centers in the arcuate nucleus of the hypothalamus (ARH). The nucleus of the solitary tract (NTS) in the brainstem has also been implicated as an important nucleus involved in appetite regulation. We set out to determine whether the NTS may be involved in inflammation-associated anorexia by injecting IL-1 beta into the 4th ventricle and assessing food intake and NTS neuronal activation. Injection of IL-1 beta produced a decrease in food intake at 3 and 12h after injection which was ameliorated at the 12h time point by a sub-threshold dose of agouti-related peptide (AgRP). Investigation into neuron types in the NTS revealed that IL-1 beta injection was associated with an increase in c-Fos activity in NTS neurons expressing tyrosine hydroxylase (TH). Additionally, injection of IL-1 beta into the 4th ventricle did not produce c-Fos activation of neurons expressing pro-opiomelanocortin (POMC) in the ARH, cells known to be involved in producing anorexia in response to systemic inflammation. Double-label in situ hybridization revealed that TH neurons did not express IL-1 receptor I (IL1-RI) transcript, demonstrating that c-Fos activation of TH neurons in this setting was not via direct stimulation of IL-1 beta on TH neurons themselves. We conclude that IL-1 beta injection into the 4th ventricle produces anorexia and is accompanied by an increase in activation in TH neurons in the NTS. This provides evidence that the brainstem may be an important mediator of anorexia in the setting of inflammation.

Agouti-related protein (AgRP) is an orexigenic neuropeptide produced by neurons in the hypothalamic arcuate nucleus (ARC) that is a key component of central neural circuits that control food intake and energy expenditure. Disorders in energy homeostasis, characterized by hypophagia and increased metabolic rate, frequently develop in animals with either acute or chronic diseases. Recently, studies have demonstrated that proopiomelanocortin-expressing neurons in the ARC are activated by the proinflammatory cytokine IL-1beta. In the current study, we sought to determine whether inflammatory processes regulate the expression of AgRP mRNA and to characterize the response of AgRP neurons to IL-1beta. Here, we show by real-time RT-PCR and in situ hybridization analysis that AgRP mRNA expression in rodents is increased in models of acute and chronic inflammation. AgRP neurons were found to express the type I IL-1 receptor, and the percentage of expression was significantly increased after peripheral administration of lipopolysaccharide. Furthermore, we demonstrate that IL-1beta inhibits the release of AgRP from hypothalamic explants. Collectively, these data indicate that proinflammatory signals decrease the secretion of AgRP while increasing the transcription of the AgRP gene. These observations suggest that AgRP neurons may participate with ARC proopiomelanocortin neurons in mediating the anorexic and metabolic responses to acute and chronic disease processes.

Background Steroid-induced sleep disturbance is a common and highly distressing morbidity for children receiving steroid chemotherapy for the treatment of pediatric acute lymphoblastic leukemia (ALL). Sleep disturbance can negatively impact overall quality of life, neurodevelopment, memory consolidation, and wound healing. Hypothalamic orexin neurons are influential wake-promoting neurons, and disturbances in orexin signaling leads to abnormal sleep behavior. A new class of drug, the orexin receptor antagonists, could be an intriguing option for sleep disorders caused by increased orexinergic output. Our aim was to examine the impact of ALL treatment doses of corticosteroids on the orexin system in rodents and in children undergoing treatment for childhood ALL. Methods We administered repeated injections of dexamethasone to rodents and measured responsive orexin neural activity compared to controls. In children with newly diagnosed standard risk B-cell ALL receiving dexamethasone therapy per Children’s Oncology Group (COG) induction therapy from 2014–2016, we collected pre- and during-steroids matched CSF samples and measured the impact of steroids on CSF orexin concentration. Results In both rodents, all markers orexin signaling, including orexin neural output and orexin receptor expression, were preserved in the setting of dexamethasone. Additionally, we did not detect a difference in pre- and during-dexamethasone CSF orexin concentrations in children receiving dexamethasone. Conclusions Our results demonstrate that rodent and human orexin physiology is largely preserved in the setting of high dose dexamethasone. The data obtained in our experimental model fail to demonstrate a causative role for disruption of the orexin pathway in steroid-induced sleep disturbance.

Metabolic syndrome, a complex of highly debilitating disorders that includes insulin resistance, hypertension, and dyslipidemia, is associated with the development of obesity in humans as well as rodent models. White adipose tissue (WAT) inflammation, caused in part by macrophage infiltration, and fat accumulation in the liver are both linked to development of the metabolic syndrome. Despite large increases in body fat, melanocortin 3-receptor (MC3-R)-deficient mice do not get fatty liver disease or severe insulin resistance. This is in contrast to obese melanocortin 4-receptor (MC4-R)-deficient mice and diet-induced obese (DIO) mice, which show increased adiposity, fatty liver disease, and insulin resistance. We hypothesized that defects in the inflammatory response to obesity may underlie the protection from metabolic syndrome seen in MC3-R null mice. MC4-R mice fed a chow diet show increased proinflammatory gene expression and macrophage infiltration in WAT, as do wild-type (WT) DIO mice. In contrast, MC3-R-deficient mice fed a normal chow diet show neither of these inflammatory changes, despite their elevated adiposity and a comparable degree of adipocyte hypertrophy to the MC4-R null and DIO mice. Furthermore, even when challenged with high-fat chow for 4 wk, a period of time shown to induce an inflammatory response in WAT of WT animals, MC3-R nulls showed an attenuated up-regulation in both monocyte chemoattractant protein-1 (MCP-1) and TNFα mRNA in WAT compared with WT high-fat-fed animals.

Chronic kidney disease (CKD) is associated with an increase in inflammatory cytokines and can result in cachexia with loss of muscle and fat stores. We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a ghrelin receptor agonist. Currently, we examine a surgical model of CKD in rats, resulting in uremia and decreased accrual of lean body mass. Treatment with ghrelin and two ghrelin receptor agonists (BIM-28125 and BIM-28131) resulted in increased food intake and an improvement in lean body mass accrual that was related in part to a decrease in muscle protein degradation as assessed by muscle levels of the 14-kDa actin fragment resulting from cleaved actomyosin. Additionally, there was a decrease in circulating inflammatory cytokines in nephrectomized animals treated with ghrelin relative to saline treatment. Ghrelin-treated animals also had a decrease in the expression of IL-1 receptor in the brainstem and a decrease in expression of prohormone convertase-2, an enzyme involved in the processing of proopiomelanocortin to the anorexigenic peptide α-MSH. We conclude that ghrelin treatment in uremia results in improved lean mass accrual in part due to suppressed muscle proteolysis and possibly related to antiinflammatory effects.

Anorexia and involuntary weight loss are common and debilitating complications of a number of chronic diseases and inflammatory states. Proinflammatory cytokines, including IL-1 beta, are hypothesized to mediate these responses through direct actions on the central nervous system. However, the neural circuits through which proinflammatory cytokines regulate food intake and energy balance remain to be characterized. Here we report that IL-1 beta activates the central melanocortin system, a key neuronal circuit in the regulation of energy homeostasis. Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) were found to express the type I IL-1 receptor. Intracerebroventricular injection of IL-1 beta induced the expression of Fos protein in ARC POMC neurons but not in POMC neurons in the commissural nucleus of the tractus solitarius. We further show that IL-1 beta increases the frequency of action potentials of ARC POMC neurons and stimulates the release of alpha-MSH from hypothalamic explants in a dose-dependent fashion. Collectively, our data support a model in which IL-1 beta increases central melanocortin signaling by activating a subpopulation of hypothalamic POMC neurons and stimulating their release of alpha-MSH.

Ras homolog enriched in brain (RHEB1) is a member within the superfamily of GTP-binding proteins encoded by the RAS oncogenes. RHEB1 is located at the crossroad of several important pathways including the insulin-signaling pathways and thus plays an important role in different physiological processes. To understand better the physiological relevance of RHEB1 protein, the expression pattern of RHEB1 was analyzed in both embryonic (at E3.5–E16.5) and adult (1-month old) mice. RHEB1 immunostaining and X-gal staining were used for wild-type and Rheb1 gene trap mutant mice, respectively. These independent methods revealed similar RHEB1 expression patterns during both embryonic and postnatal developments. Ubiquitous uniform RHEB1/β-gal and/or RHEB1 expression was seen in preimplantation embryos at E3.5 and postimplantation embryos up to E12.5. Between stages E13.5 and E16.5, RHEB1 expression levels became complex: In particular, strong expression was identified in neural tissues, including the neuroepithelial layer of the mesencephalon, telencephalon, and neural tube of CNS and dorsal root ganglia. In addition, strong expression was seen in certain peripheral tissues including heart, intestine, muscle, and urinary bladder. Postnatal mice have broad spatial RHEB1 expression in different regions of the cerebral cortex, subcortical regions (including hippocampus), olfactory bulb, medulla oblongata, and cerebellum (particularly in Purkinje cells). Significant RHEB1 expression was also viewed in internal organs including the heart, intestine, urinary bladder, and muscle. Moreover, adult animals have complex tissue- and organ-specific RHEB1 expression patterns with different intensities observed throughout postnatal development. Its expression level is in general comparable in CNS and other organs of mouse. Thus, the expression pattern of RHEB1 suggests that it likely plays a ubiquitous role in the development of the early embryo with more tissue-specific roles in later development.

When challenged with a variety of inflammatory threats, multiple systems across the body undergo physiological responses to promote defense and survival. The constellation of fever, anorexia, and fatigue is known as the acute illness response, and represents an adaptive behavioral and physiological reaction to stimuli such as infection. On the other end of the spectrum, cachexia is a deadly and clinically challenging syndrome involving anorexia, fatigue, and muscle wasting. Both of these processes are governed by inflammatory mediators including cytokines, chemokines, and immune cells. Though the effects of cachexia can be partially explained by direct effects of disease processes on wasting tissues, a growing body of evidence shows the central nervous system (CNS) also plays an essential mechanistic role in cachexia. In the context of inflammatory stress, the hypothalamus integrates signals from peripheral systems, which it translates into neuroendocrine perturbations, altered neuronal signaling, and global metabolic derangements. Therefore, we will discuss how hypothalamic inflammation is an essential driver of both the acute illness response and cachexia, and why this organ is uniquely equipped to generate and maintain chronic inflammation. First, we will focus on the role of the hypothalamus in acute responses to dietary and infectious stimuli. Next, we will discuss the role of cytokines in driving homeostatic disequilibrium, resulting in muscle wasting, anorexia, and weight loss. Finally, we will address mechanisms and mediators of chronic hypothalamic inflammation, including endothelial cells, chemokines, and peripheral leukocytes.