Restoring SRSF3 in Kupffer cells attenuates obesity-related insulin resistance.

BACKGROUND AND AIMS: In obesity, depletion of KCs expressing CRIg (complement receptor of the Ig superfamily) leads to microbial DNA accumulation, which subsequently triggers tissue inflammation and insulin resistance. However, the mechanism underlying obesity-mediated changes in KC complement immune functions is largely unknown. APPROACH AND RESULTS: Using KC-specific deactivated Cas9 transgenic mice treated with guide RNA, we assessed the effects of restoring CRIg or the serine/arginine-rich splicing factor 3 (SRSF3) abundance on KC functions and metabolic phenotypes in obese mice. The impacts of weight loss on KC responses were evaluated in a diet switch mouse model. The role of SRSF3 in regulating KC functions was also evaluated using KC-specific SRSF3 knockout mice. Here, we report that overexpression of CRIg in KCs of obese mice protects against bacterial DNA accumulation in metabolic tissues. Mechanistically, SRSF3 regulates CRIg expression, which is essential for maintaining the CRIg+ KC population. During obesity, SRSF3 expression decreases, but it is restored with weight loss through a diet switch, normalizing CRIg+ KCs. KC SRSF3 is also repressed in obese human livers. Lack of SRSF3 in KCs in lean and obese mice decreases their CRIg+ population, impairing metabolic parameters. During the diet switch, the benefits of weight loss are compromised due to SRSF3 deficiency. Conversely, SRSF3 overexpression in obese mice preserves CRIg+ KCs and improves metabolic responses. CONCLUSIONS: Restoring SRSF3 abundance in KCs offers a strategy against obesity-associated tissue inflammation and insulin resistance by preventing bacterial DNA accumulation.

Testicular germ cell tumors arise in the absence of sex-specific differentiation.

In response to signals from the embryonic testis, the germ cell intrinsic factor NANOS2 coordinates a transcriptional program necessary for the differentiation of pluripotent-like primordial germ cells toward a unipotent spermatogonial stem cell fate. Emerging evidence indicates that genetic risk factors contribute to testicular germ cell tumor initiation by disrupting sex-specific differentiation. Here, using the 129.MOLF-Chr19 mouse model of testicular teratomas and a NANOS2 reporter allele, we report that the developmental phenotypes required for tumorigenesis, including failure to enter mitotic arrest, retention of pluripotency and delayed sex-specific differentiation, were exclusive to a subpopulation of germ cells failing to express NANOS2. Single-cell RNA sequencing revealed that embryonic day 15.5 NANOS2-deficient germ cells and embryonal carcinoma cells developed a transcriptional profile enriched for MYC signaling, NODAL signaling and primed pluripotency. Moreover, lineage-tracing experiments demonstrated that embryonal carcinoma cells arose exclusively from germ cells failing to express NANOS2. Our results indicate that NANOS2 is the nexus through which several genetic risk factors influence tumor susceptibility. We propose that, in the absence of sex specification, signals native to the developing testis drive germ cell transformation.

Obesity, cancer risk, and time-restricted eating.

Obesity and the associated metabolic syndrome is considered a pandemic whose prevalence is steadily increasing in many countries worldwide. It is a complex, dynamic, and multifactorial disorder that presages the development of several metabolic, cardiovascular, and neurodegenerative diseases, and increases the risk of cancer. In patients with newly diagnosed cancer, obesity worsens prognosis, increasing the risk of recurrence and decreasing survival. The multiple negative effects of obesity on cancer outcomes are substantial, and of great clinical importance. Strategies for weight control have potential utility for both prevention efforts and enhancing cancer outcomes. Presently, time-restricted eating (TRE) is a popular dietary intervention that involves limiting the consumption of calories to a specific window of time without any proscribed caloric restriction or alteration in dietary composition. As such, TRE is a sustainable long-term behavioral modification, when compared to other dietary interventions, and has shown many health benefits in animals and humans. The preliminary data regarding the effects of time-restricted feeding on cancer development and growth in animal models are promising but studies in humans are lacking. Interestingly, several short-term randomized clinical trials of TRE have shown favorable effects to reduce cancer risk factors; however, long-term trials of TRE have yet to investigate reductions in cancer incidence or outcomes in the general population. Few studies have been conducted in cancer populations, but a number are underway to examine the effect of TRE on cancer biology and recurrence. Given the simplicity, feasibility, and favorable metabolic improvements elicited by TRE in obese men and women, TRE may be useful in obese cancer patients and cancer survivors; however, the clinical implementation of TRE in the cancer setting will require greater in-depth investigation.

Treatment of Hepatocellular Carcinoma by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant.

PURPOSE: To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Mice with bilateral, subcutaneous RIL-175 cell-derived HCCs were randomized to 4 groups: (a) phosphate-buffered saline (control), (b) cryoablation only (Cryo), (c) CPMV-treated only (CPMV), and (d) cryoablation plus CPMV-treated (Cryo + CPMV) (N = 11-14 per group). Intratumoral CPMV was administered every 3 days for 4 doses, with cryoablation performed on the third day. Contralateral tumors were monitored. Tumor growth and systemic chemokine/cytokine levels were measured. A subset of tumors and spleens were harvested for immunohistochemistry (IHC) and flow cytometry. One- or 2-way analysis of variance was performed for statistical comparisons. A P value of <.05 was used as the threshold for statistical significance. RESULTS: At 2 weeks after treatment, the Cryo and CPMV groups, alone or combined, outperformed the control group in the treated tumor; however, the Cryo + CPMV group showed the strongest reduction and lowest variance (1.6-fold ± 0.9 vs 6.3-fold ± 0.5, P < .0001). For the untreated tumor, only Cryo + CPMV significantly reduced tumor growth compared with control (9.2-fold ± 0.9 vs 17.8-fold ± 2.1, P = .01). The Cryo + CPMV group exhibited a transient increase in interleukin-10 and persistently decreased CXCL1. Flow cytometry revealed natural killer cell enrichment in the untreated tumor and increased PD-1 expression in the spleen. Tumor-infiltrating lymphocytes increased in Cryo + CPMV-treated tumors by IHC. CONCLUSIONS: Cryoablation and intratumoral CPMV, alone or combined, demonstrated potent efficacy against treated HCC tumors; however, only cryoablation combined with CPMV slowed the growth of untreated tumors, consistent with an abscopal effect.

Delayed male germ cell sex-specification permits transition into embryonal carcinoma cells with features of primed pluripotency.

Testicular teratomas result from anomalies in embryonic germ cell development. In 129 inbred mice, teratoma initiation coincides with germ cell sex-specific differentiation and the mitotic-meiotic switch: XX and XY germ cells repress pluripotency, XX germ cells initiate meiosis, and XY germ cells activate male-specific differentiation and mitotic arrest. Here, we report that expression of Nanos2, a gene that is crucial to male sex specification, is delayed in teratoma-susceptible germ cells. Decreased expression of Nanos2 was found to be due, in part, to the Nanos2 allele present in 129 mice. In teratoma-susceptible germ cells, diminished expression of genes downstream of Nanos2 disrupted processes that were crucial to male germ cell differentiation. Deficiency for Nanos2 increased teratoma incidence in 129 mice and induced developmental abnormalities associated with tumor initiation in teratoma-resistant germ cells. Finally, in the absence of commitment to the male germ cell fate, we discovered that a subpopulation of teratoma-susceptible germ cells transition into embryonal carcinoma (EC) cells with primed pluripotent features. We conclude that delayed male germ cell sex-specification facilitates the transformation of germ cells with naïve pluripotent features into primed pluripotent EC cells.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 µM) and PACAP (0.1 µM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 µM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

Cyclic AMP concentrations in dendritic cells induce and regulate Th2 immunity and allergic asthma.

The inductive role of dendritic cells (DC) in Th2 differentiation has not been fully defined. We addressed this gap in knowledge by focusing on signaling events mediated by the heterotrimeric GTP binding proteins Gαs, and Gαi, which respectively stimulate and inhibit the activation of adenylyl cyclases and the synthesis of cAMP. We show here that deletion of Gnas, the gene that encodes Gαs in mouse CD11c(+) cells (Gnas(ΔCD11c) mice), and the accompanying decrease in cAMP provoke Th2 polarization and yields a prominent allergic phenotype, whereas increases in cAMP inhibit these responses. The effects of cAMP on DC can be demonstrated in vitro and in vivo and are mediated via PKA. Certain gene products made by Gnas(ΔCD11c) DC affect the Th2 bias. These findings imply that G protein-coupled receptors, the physiological regulators of Gαs and Gαi activation and cAMP formation, act via PKA to regulate Th bias in DC and in turn, Th2-mediated immunopathologies.

Chromogranin A regulates vesicle storage and mitochondrial dynamics to influence insulin secretion.

Chromogranin A (CgA) is a prohormone and a granulogenic factor that regulates secretory pathways in neuroendocrine tissues. In ß-cells of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. The impact of CgA deficiency on the formation and exocytosis of insulin vesicles is yet to be investigated. In addition, no literature exists on the impact of CgA on mitochondrial function in ß-cells. Using three different antibodies, we demonstrate that CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. CgA deficiency in Chga-KO islets leads to compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins and insulin genes, as well as increased insulin protein content. Ultrastructural studies of pancreatic islets revealed that Chga-KO ß-cells contain fewer immature secretory granules than wild-type (WT) control but increased numbers of mature secretory granules and plasma membrane-docked vesicles. Compared to WT control, CgA-deficient ß-cells exhibited increases in mitochondrial volume, numerical densities and fusion, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1 and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion observed in Chga-KO mice. We conclude that CgA is an important regulator for coordination of mitochondrial dynamics, secretory vesicular quanta and GSIS for optimal secretory functioning of ß-cells, suggesting a strong, CgA-dependent positive link between mitochondrial fusion and GSIS.

Deletion of serine/arginine-rich splicing factor 3 in hepatocytes predisposes to hepatocellular carcinoma in mice.

UNLABELLED: Alterations in RNA splicing are associated with cancer, but it is not clear whether they result from malignant transformation or have a causative role. We show here that hepatocyte-specific deletion of serine/arginine-rich splicing factor 3 (SRSF3) impairs hepatocyte maturation and metabolism in early adult life, and mice develop spontaneous hepatocellular carcinoma (HCC) with aging. Tumor development is preceded by chronic liver disease with progressive steatosis and fibrosis. SRSF3 protects mice against CCl4 -induced fibrosis and carcinogenesis and suppresses inclusion of the profibrogenic EDA exon in fibronectin 1. Loss of SRSF3 increases expression of insulin-like growth factor 2 and the A-isoform of the insulin receptor, allowing aberrant activation of mitogenic signaling, promotes aberrant splicing and expression of epithelial to mesenchymal transition (EMT) genes, and activates Wnt/ß-catenin signaling leading to c-Myc induction. Finally, SRSF3 expression is either decreased or the protein mislocalized in human HCC. CONCLUSION: Our data suggest a potential role for SRSF3 in preventing hepatic carcinogenesis by regulating splicing to suppress fibrosis, mitogenic splicing, and EMT. Thus, these mice may provide an attractive model to discover the pathogenic mechanisms linking aberrant pre-messenger RNA splicing with liver damage, fibrosis, and HCC.

The transcriptome of a complete episode of acute otitis media.

BACKGROUND: Otitis media is the most common disease of childhood, and represents an important health challenge to the 10-15% of children who experience chronic/recurrent middle ear infections. The middle ear undergoes extensive modifications during otitis media, potentially involving changes in the expression of many genes. Expression profiling offers an opportunity to discover novel genes and pathways involved in this common childhood disease. The middle ears of 320 WBxB6 F1 hybrid mice were inoculated with non-typeable Haemophilus influenzae (NTHi) or PBS (sham control). Two independent samples were generated for each time point and condition, from initiation of infection to resolution. RNA was profiled on Affymetrix mouse 430 2.0 whole-genome microarrays. RESULTS: Approximately 8% of the sampled transcripts defined the signature of acute NTHi-induced otitis media across time. Hierarchical clustering of signal intensities revealed several temporal gene clusters. Network and pathway enrichment analysis of these clusters identified sets of genes involved in activation of the innate immune response, negative regulation of immune response, changes in epithelial and stromal cell markers, and the recruitment/function of neutrophils and macrophages. We also identified key transcriptional regulators related to events in otitis media, which likely determine the expression of these gene clusters. A list of otitis media susceptibility genes, derived from genome-wide association and candidate gene studies, was significantly enriched during the early induction phase and the middle re-modeling phase of otitis but not in the resolution phase. Our results further indicate that positive versus negative regulation of inflammatory processes occur with highly similar kinetics during otitis media, underscoring the importance of anti-inflammatory responses in controlling pathogenesis. CONCLUSIONS: The results characterize the global gene response during otitis media and identify key signaling and transcription factor networks that control the defense of the middle ear against infection. These networks deserve further attention, as dysregulated immune defense and inflammatory responses may contribute to recurrent or chronic otitis in children.

A Novel Letrozole Model Recapitulates Both the Reproductive and Metabolic Phenotypes of Polycystic Ovary Syndrome in Female Mice.

Polycystic ovary syndrome (PCOS) pathophysiology is poorly understood, due partly to lack of PCOS animal models fully recapitulating this complex disorder. Recently, a PCOS rat model using letrozole (LET), a nonsteroidal aromatase inhibitor, mimicked multiple PCOS phenotypes, including metabolic features absent in other models. Given the advantages of using genetic and transgenic mouse models, we investigated whether LET produces a similar PCOS phenotype in mice. Pubertal female C57BL/6N mice were treated for 5 wk with LET, which resulted in increased serum testosterone and normal diestrus levels of estradiol, similar to the hyperandrogenemia and follicular phase estrogen levels of PCOS women. As in PCOS, ovaries from LET mice were larger, polycystic, and lacked corpora lutea versus controls. Most LET females were acyclic, and all were infertile. LET females displayed elevated serum LH levels and higher Lhb mRNA in the pituitary. In contrast, serum FSH and Fshb were significantly reduced in LET females, demonstrating differential effects on gonadotropins, as in PCOS. Within the ovary, LET females had higher Cyp17, Cyp19, and Fsh receptor mRNA expression. In the hypothalamus, LET females had higher kisspeptin receptor mRNA expression but lower progesterone receptor mRNA levels. LET females also gained more weight than controls, had increased abdominal adiposity and adipocyte size, elevated adipose inflammatory mRNA levels, and impaired glucose tolerance, mirroring the metabolic phenotype in PCOS women. This is the first report of a LET paradigm in mice that recapitulates both reproductive and metabolic PCOS phenotypes and will be useful to genetically probe the PCOS condition.

Molecular tumor board: the University of California-San Diego Moores Cancer Center experience.

OBJECTIVE: DNA sequencing tests are enabling physicians to interrogate the molecular profiles of patients' tumors, but most oncologists have not been trained in advanced genomics. We initiated a molecular tumor board to provide expert multidisciplinary input for these patients. MATERIALS AND METHODS: A team that included clinicians, basic scientists, geneticists, and bioinformatics/pathway scientists with expertise in various cancer types attended. Molecular tests were performed in a Clinical Laboratory Improvement Amendments environment. RESULTS: Patients (n = 34, since December 2012) had received a median of three prior therapies. The median time from physician order to receipt of molecular diagnostic test results was 27 days (range: 14-77 days). Patients had a median of 4 molecular abnormalities (range: 1-14 abnormalities) found by next-generation sequencing (182- or 236-gene panels). Seventy-four genes were involved, with 123 distinct abnormalities. Importantly, no two patients had the same aberrations, and 107 distinct abnormalities were seen only once. Among the 11 evaluable patients whose treatment had been informed by molecular diagnostics, 3 achieved partial responses (progression-free survival of 3.4 months, ≥6.5 months, and 7.6 months). The most common reasons for being unable to act on the molecular diagnostic results were that patients were ineligible for or could not travel to an appropriately targeted clinical trial and/or that insurance would not cover the cognate agents. CONCLUSION: Genomic sequencing is revealing complex molecular profiles that differ by patient. Multidisciplinary molecular tumor boards may help optimize management. Barriers to personalized therapy include access to appropriately targeted drugs.

Dysregulation of RNA splicing in early non-alcoholic fatty liver disease through hepatocellular carcinoma.

While changes in RNA splicing have been extensively studied in hepatocellular carcinoma (HCC), no studies have systematically investigated changes in RNA splicing during earlier liver disease. Mouse studies have shown that disruption of RNA splicing can trigger liver disease and we have shown that the splicing factor SRSF3 is decreased in the diseased human liver, so we profiled RNA splicing in liver samples from twenty-nine individuals with no-history of liver disease or varying degrees of non-alcoholic fatty liver disease (NAFLD). We compared our results with three publicly available transcriptome datasets that we re-analyzed for splicing events (SEs). We found many changes in SEs occurred during early liver disease, with fewer events occurring with the onset of inflammation and fibrosis. Many of these early SEs were enriched for SRSF3-dependent events and were associated with SRSF3 binding sites. Mapping the early and late changes to gene ontologies and pathways showed that the genes harboring these early SEs were involved in normal liver metabolism, whereas those harboring late SEs were involved in inflammation, fibrosis and proliferation. We compared the SEs with HCC data from the TCGA and observed that many of these early disease SEs are found in HCC samples and, furthermore, are correlated with disease survival. Changes in splicing factor expression are also observed, which may be associated with distinct subsets of the SEs. The maintenance of these SEs through the multi-year oncogenic process suggests that they may be causative. Understanding the role of these splice variants in metabolic liver disease progression may shed light on the triggers of liver disease progression and the pathogenesis of HCC.

Time-Restricted Feeding Attenuates Metabolic Dysfunction-Associated Steatohepatitis and Hepatocellular Carcinoma in Obese Male Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) has surpassed the hepatitis B virus and hepatitis C virus as the leading cause of chronic liver disease in most parts of the Western world. MASLD (formerly known as NAFLD) encompasses both simple steatosis and more aggressive metabolic dysfunction-associated steatohepatitis (MASH), which is accompanied by inflammation, fibrosis, and cirrhosis, and ultimately can lead to hepatocellular carcinoma (HCC). There are currently very few approved therapies for MASH. Weight loss strategies such as caloric restriction can ameliorate the harmful metabolic effect of MASH and inhibit HCC; however, it is difficult to implement and maintain in daily life, especially in individuals diagnosed with HCC. In this study, we tested a time-restricted feeding (TRF) nutritional intervention in mouse models of MASH and HCC. We show that TRF abrogated metabolic dysregulation induced by a Western diet without any calorie restriction or weight loss. TRF improved insulin sensitivity and reduced hyperinsulinemia, liver steatosis, inflammation, and fibrosis. Importantly, TRF inhibited liver tumors in two mouse models of obesity-driven HCC. Our data suggest that TRF is likely to be effective in abrogating MASH and HCC and warrant further studies of time-restricted eating in humans with MASH who are at higher risk of developing HCC.

TM7SF3 controls TEAD1 splicing to prevent MASH-induced liver fibrosis.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax.

Cholera toxin (CT) elicits a mucosal immune response in mice when used as a vaccine adjuvant. The mechanisms by which CT exerts its adjuvant effects are incompletely understood. We show that protection against inhalation anthrax by an irradiated spore vaccine depends on CT-mediated induction of IL-17-producing CD4 Th17 cells. Furthermore, IL-17 is involved in the induction of serum and mucosal antibody responses by CT. Th17 cells induced by CT have a unique cytokine profile compared with those induced by IL-6 and TGF-beta, and their induction by CT requires cAMP-dependent secretion of IL-1beta and beta-calcitonin gene-related peptide by dendritic cells. These findings demonstrate that Th17 cells mediate mucosal adjuvant effects of CT and identify previously unexplored pathways involved in Th17 induction that could be targeted for development of unique mucosal adjuvants.

Loss of cAMP Signaling in CD11c Immune Cells Protects Against Diet-Induced Obesity.

In obesity, CD11c+ innate immune cells are recruited to adipose tissue and create an inflammatory state that causes both insulin and catecholamine resistance. We found that ablation of Gnas, the gene that encodes Gαs, in CD11c expressing cells protects mice from obesity, glucose intolerance, and insulin resistance. Transplantation studies showed that the lean phenotype was conferred by bone marrow-derived cells and did not require adaptive immunity. Loss of cAMP signaling was associated with increased adipose tissue norepinephrine and cAMP signaling, and prevention of catecholamine resistance. The adipose tissue had reduced expression of catecholamine transport and degradation enzymes, suggesting that the elevated norepinephrine resulted from decreased catabolism. Collectively, our results identified an important role for cAMP signaling in CD11c+ innate immune cells in whole-body metabolism by controlling norepinephrine levels in white adipose tissue, modulating catecholamine-induced lipolysis and increasing thermogenesis, which, together, created a lean phenotype. ARTICLE HIGHLIGHTS: We undertook this study to understand how immune cells communicate with adipocytes, specifically, whether cAMP signaling in the immune cell and the adipocyte are connected. We identified a reciprocal interaction between CD11c+ innate immune cells and adipocytes in which high cAMP signaling in the immune cell compartment induces low cAMP signaling in adipocytes and vice versa. This interaction regulates lipolysis in adipocytes and inflammation in immune cells, resulting in either a lean, obesity-resistant, and insulin-sensitive phenotype, or an obese, insulin-resistant phenotype.

Long-term exposure to house dust mites accelerates lung cancer development in mice.

BACKGROUND: Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1ß (IL-1ß), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1ß signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1ß, and CCL2 neutralization, or ICS treatment. CONCLUSIONS: Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

Catestatin induces glycogenesis by stimulating the phosphoinositide 3-kinase-AKT pathway.

AIM: Defects in hepatic glycogen synthesis contribute to post-prandial hyperglycaemia in type 2 diabetic patients. Chromogranin A (CgA) peptide Catestatin (CST: hCgA352-372 ) improves glucose tolerance in insulin-resistant mice. Here, we seek to determine whether CST induces hepatic glycogen synthesis. METHODS: We determined liver glycogen, glucose-6-phosphate (G6P), uridine diphosphate glucose (UDPG) and glycogen synthase (GYS2) activities; plasma insulin, glucagon, noradrenaline and adrenaline levels in wild-type (WT) as well as in CST knockout (CST-KO) mice; glycogen synthesis and glycogenolysis in primary hepatocytes. We also analysed phosphorylation signals of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-dependent kinase-1 (PDK-1), GYS2, glycogen synthase kinase-3ß (GSK-3ß), AKT (a kinase in AKR mouse that produces Thymoma)/PKB (protein kinase B) and mammalian/mechanistic target of rapamycin (mTOR) by immunoblotting. RESULTS: CST stimulated glycogen accumulation in fed and fasted liver and in primary hepatocytes. CST reduced plasma noradrenaline and adrenaline levels. CST also directly stimulated glycogenesis and inhibited noradrenaline and adrenaline-induced glycogenolysis in hepatocytes. In addition, CST elevated the levels of UDPG and increased GYS2 activity. CST-KO mice had decreased liver glycogen that was restored by treatment with CST, reinforcing the crucial role of CST in hepatic glycogenesis. CST improved insulin signals downstream of IR and IRS-1 by enhancing phospho-AKT signals through the stimulation of PDK-1 and mTORC2 (mTOR Complex 2, rapamycin-insensitive complex) activities. CONCLUSIONS: CST directly promotes the glycogenic pathway by (a) reducing glucose production, (b) increasing glycogen synthesis from UDPG, (c) reducing glycogenolysis and (d) enhancing downstream insulin signalling.

The ECRG4 cleavage product augurin binds the endotoxin receptor and influences the innate immune response during otitis media.

Otitis media (OM), the most common disease of childhood, is typically characterized by bacterial infection of the middle ear (ME). Prominent features of OM include hyperplasia of the ME mucosa, which transforms from a monolayer of simple squamous epithelium with minimal stroma into a full-thickness respiratory epithelium in 2-3 days after infection. Analysis of the murine ME transcriptome during OM showed down-regulation of the tumor suppressor gene Ecrg4 that was temporally related to mucosal hyperplasia and identified stromal cells as the primary ECRG4 source. The reduction in Ecrg4 gene expression coincided with the cleavage of ECRG4 protein to release an extracellular fragment, augurin. The duration of mucosal hyperplasia during OM was greater in Ecrg4 -/- mice, the number of infiltrating macrophages was enhanced, and ME infection cleared more rapidly. ECRG4-null macrophages showed increased bacterial phagocytosis. Co-immunoprecipitation identified an association of augurin with TLR4, CD14 and MD2, the components of the lipopolysaccharide (LPS) receptor. The results suggest that full-length ECRG4 is a sentinel molecule that potentially inhibits growth of the ME stroma. Processing of ECRG4 protein during inflammation, coupled with a decline in Ecrg4 gene expression, also influences the behavior of cells that do not express the gene, limiting the production of growth factors by epithelial and endothelial cells, as well as the activity of macrophages.