Calcium-dependent protein kinase GhCDPK4 plays a role in drought and abscisic acid stress responses.

Drought is an important factor limiting the yield and quality of cotton. In the present study, the gene encoding the cotton calcium-dependent protein kinase GhCDPK4 was identified and characterized in the transcriptome of cotton under PEG-induced drought stress. In RT-qPCR experiments, GhCDPK4 expression was found to be up-regulated under drought and abscisic acid (ABA) stress. Under drought conditions, heterologous overexpression of GhCDPK4 in tobacco showed a better phenotypic status, higher antioxidant enzyme activity, and lower relative electrolyte leakage (REL) and malondialdehyde (MDA) content. Meanwhile, ghcdpk4-silenced cotton plants, which were extremely sensitive to drought, exhibited higher levels of O2-，H2O2, and MDA contents compared to the control. Meanwhile, silenced lines showed impaired stomatal closure under drought stress, resulting in increased water loss from transpiration in silenced lines. GhCDPK4 expression was induced by ABA, and there are five ABA-responsive elements in its promoter. and C2-DOMAIN ABA-RELATED 4(CAR4, Gh_D09G1653) were found to interact and be co-expressed in the GhCDPK4 interaction network. Therefore, GhCDPK4 may reduce the extent of water loss and oxidative damage in cotton under drought by positively regulating ABA-controlled stomatal closure and reactive oxygen species (ROS) scavenging systems. This study demonstrates the great potential of GhCDPK4 in improving drought resistance in crops.

Lipopolysaccharide-induced hypothalamic inflammation in cancer cachexia-anorexia is amplified by tumour-derived prostaglandin E2.

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.

Nanoparticle-Based Follistatin Messenger RNA Therapy for Reprogramming Metastatic Ovarian Cancer and Ameliorating Cancer-Associated Cachexia.

This study presents the first messenger RNA (mRNA) therapy for metastatic ovarian cancer and cachexia-induced muscle wasting based on lipid nanoparticles that deliver follistatin (FST) mRNA predominantly to cancer clusters following intraperitoneal administration. The secreted FST protein, endogenously synthesized from delivered mRNA, efficiently reduces elevated activin A levels associated with aggressive ovarian cancer and associated cachexia. By altering the cancer cell phenotype, mRNA treatment prevents malignant ascites, delays cancer progression, induces the formation of solid tumors, and preserves muscle mass in cancer-bearing mice by inhibiting negative regulators of muscle mass. Finally, mRNA therapy provides synergistic effects in combination with cisplatin, increasing the survival of mice and counteracting muscle atrophy induced by chemotherapy and cancer-associated cachexia. The treated mice develop few nonadherent tumors that are easily resected from the peritoneum. Clinically, this nanomedicine-based mRNA therapy can facilitate complete cytoreduction, target resistance, improve resilience during aggressive chemotherapy, and improve survival in advanced ovarian cancer.

Physiologic and molecular characterization of a novel murine model of metastatic head and neck cancer cachexia.

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 < 0.05). Behaviourally, MLM3-implanted mice displayed decreased locomotor behaviours and impaired nest building (P < 0.05). Muscle catabolism programmes associated with cachexia, including E3 ubiquitin ligase and autophagy up-regulation, along with progressive adipose wasting and accompanying browning gene signatures, were observed. Tumour progression also corresponded with hypothalamic and peripheral organ inflammation, as well as an elevation in neutrophil-to-lymphocyte ratio (P < 0.05). Finally, we characterize the fat and lean mass sparing effects of voluntary wheel running on MLM3 cachexia (P < 0.05). CONCLUSIONS: This syngeneic MLM3 allograft model of metastatic cancer cachexia is reliable, consistent, and readily recapitulates key clinical and molecular features and heterogeneity of cancer cachexia. Because few metastatic models of cachexia exist-even though cachexia often accompanies metastatic progression-we believe this model more accurately captures cancer cachexia observed in a clinical setting and thus is well suited for future mechanistic studies and pre-clinical therapy development for this crippling metabolic disorder.

Chronic cerebral lipocalin 2 exposure elicits hippocampal neuronal dysfunction and cognitive impairment.

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.

Lipocalin 2 mediates appetite suppression during pancreatic cancer cachexia.

Lipocalin 2 (LCN2) was recently identified as an endogenous ligand of the type 4 melanocortin receptor (MC4R), a critical regulator of appetite. However, it remains unknown if this molecule influences appetite during cancer cachexia, a devastating clinical entity characterized by decreased nutrition and progressive wasting. We demonstrate that LCN2 is robustly upregulated in murine models of pancreatic cancer, its expression is associated with reduced food consumption, and Lcn2 deletion is protective from cachexia-anorexia. Consistent with LCN2's proposed MC4R-dependent role in cancer-induced anorexia, pharmacologic MC4R antagonism mitigates cachexia-anorexia, while restoration of Lcn2 expression in the bone marrow is sufficient in restoring the anorexia feature of cachexia. Finally, we observe that LCN2 levels correlate with fat and lean mass wasting and is associated with increased mortality in patients with pancreatic cancer. Taken together, these findings implicate LCN2 as a pathologic mediator of appetite suppression during pancreatic cancer cachexia.

Melanocortin-4 receptor antagonist TCMCB07 ameliorates cancer- and chronic kidney disease-associated cachexia.

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.

Association of Sarcopenia With Oncologic Outcomes of Primary Surgery or Definitive Radiotherapy Among Patients With Localized Oropharyngeal Squamous Cell Carcinoma.

Importance: The negative association of low lean muscle mass (sarcopenia) with survival outcomes in head and neck cancers, including oropharyngeal carcinoma, is established. However, it is not known whether the choice of primary treatment modality (surgery or radiotherapy) is associated with oncologic outcomes of patients with sarcopenia and oropharyngeal squamous cell carcinoma (OPSCC). Objective: To examine whether primary surgical resection or definitive radiotherapy is associated with improved survival for patients with sarcopenia and localized OPSCC. Design, Setting, and Participants: A cohort study was conducted of patients with clinically staged T1 to T2, N0 to N2 OPSCC with cross-sectional abdominal imaging within 60 days prior to treatment and treated between January 1, 2005, and December 31, 2017. Skeletal muscle mass was measured at the third lumbar vertebra using previously defined techniques and sarcopenia was defined as less than 52.4 cm2/m2 of muscle for men and less than 38.5 cm2/m2 for women. In addition, associated patient demographic characteristics, cancer data, treatment information, and survival outcomes were assessed. Statistical analysis was performed from December 3, 2018, to August 28, 2019. Main Outcomes and Measures: Primary outcomes were overall survival and disease-specific survival. Results: Among the 245 patients who met study inclusion criteria, 209 were men (85.3%) and the mean (SD) age was 62.3 (7.8) years. Sarcopenia was detected in 135 patients (55.1%), while normal skeletal muscle mass was detected in 110 patients (44.9%). For the 110 patients without sarcopenia, primary treatment modality was not associated with improved survival. For patients with sarcopenia at diagnosis, primary surgical resection was associated with improved overall survival (hazard ratio [HR], 0.37; 95% CI, 0.17-0.82) and disease-specific survival (HR, 0.22; 95% CI, 0.07-0.68). This association persisted after propensity score matching, as up-front surgery was associated with improved overall survival (HR, 0.33; 95% CI, 0.12-0.91) and disease-specific survival (HR, 0.17; 95% CI, 0.04-0.75) survival. Conclusions and Relevance: This study suggests that sarcopenia has a negative association with survival for patients with OPSCC. Primary surgery and radiotherapy confer similar survival associations for patients with normal skeletal muscle mass and localized OPSCC. However, up-front surgical resection may be associated with improved survival outcomes for patients with sarcopenia.

Circulating myeloid cells invade the central nervous system to mediate cachexia during pancreatic cancer.

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.

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.

Microglia in the hypothalamus respond to tumor-derived factors and are protective against cachexia during pancreatic cancer.

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.

Publisher Correction: The TLR7/8 agonist R848 remodels tumor and host responses to promote survival in pancreatic cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The TLR7/8 agonist R848 remodels tumor and host responses to promote survival in pancreatic cancer.

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+ T-cell infiltration and activity, and decreased Treg frequency. R848-treated mice demonstrated improvements in behavioral and molecular cachexia manifestations, resulting in a near-doubling of survival duration. Knockout mouse studies revealed that stromal, not neoplastic, TLR7 is requisite for R848-mediated responses. In patient samples, we found Tlr7 is ubiquitously expressed in stroma across all stages of pancreatic neoplasia, but epithelial Tlr7 expression is relatively uncommon. These studies indicate immune-enhancing approaches including R848 may be useful in PDAC and cancer-associated cachexia.

MyD88 signalling is critical in the development of pancreatic cancer cachexia.

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.

TRIF is a key inflammatory mediator of acute sickness behavior and cancer cachexia.

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.

Establishment and characterization of a novel murine model of pancreatic cancer cachexia.

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.

Dexamethasone Chemotherapy Does Not Disrupt Orexin Signaling.

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.

A distinct brain pathway links viral RNA exposure to sickness behavior.

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.

Maternal high-fat diet and obesity compromise fetal hematopoiesis.

OBJECTIVE: Recent evidence indicates that the adult hematopoietic system is susceptible to diet-induced lineage skewing. It is not known whether the developing hematopoietic system is subject to metabolic programming via in utero high-fat diet (HFD) exposure, an established mechanism of adult disease in several organ systems. We previously reported substantial losses in offspring liver size with prenatal HFD. As the liver is the main hematopoietic organ in the fetus, we asked whether the developmental expansion of the hematopoietic stem and progenitor cell (HSPC) pool is compromised by prenatal HFD and/or maternal obesity. METHODS: We used quantitative assays, progenitor colony formation, flow cytometry, transplantation, and gene expression assays with a series of dietary manipulations to test the effects of gestational high-fat diet and maternal obesity on the day 14.5 fetal liver hematopoietic system. RESULTS: Maternal obesity, particularly when paired with gestational HFD, restricts physiological expansion of fetal HSPCs while promoting the opposing cell fate of differentiation. Importantly, these effects are only partially ameliorated by gestational dietary adjustments for obese dams. Competitive transplantation reveals compromised repopulation and myeloid-biased differentiation of HFD-programmed HSPCs to be a niche-dependent defect, apparent in HFD-conditioned male recipients. Fetal HSPC deficiencies coincide with perturbations in genes regulating metabolism, immune and inflammatory processes, and stress response, along with downregulation of genes critical for hematopoietic stem cell self-renewal and activation of pathways regulating cell migration. CONCLUSIONS: Our data reveal a previously unrecognized susceptibility to nutritional and metabolic developmental programming in the fetal HSPC compartment, which is a partially reversible and microenvironment-dependent defect perturbing stem and progenitor cell expansion and hematopoietic lineage commitment.

Muscle atrophy in response to cytotoxic chemotherapy is dependent on intact glucocorticoid signaling in skeletal muscle.

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.

Systemic inflammation and insulin sensitivity in obese IFN-γ knockout mice.

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.