Single-cell profiling identifies mechanisms of inflammatory heterogeneity in chronic rhinosinusitis.

The heterogeneous cellular microenvironment of human airway chronic inflammatory diseases, including chronic rhinosinusitis (CRS) and asthma, is still poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) on the nasal mucosa of healthy individuals and patients with three subtypes of CRS and identified disease-specific cell subsets and molecules that specifically contribute to the pathogenesis of CRS subtypes. As such, ALOX15+ macrophages contributed to the type 2 immunity-driven pathogenesis of one subtype of CRS, eosinophilic CRS with nasal polyps (eCRSwNP), by secreting chemokines that recruited eosinophils, monocytes and T helper 2 (TH2) cells. An inhibitor of ALOX15 reduced the release of proinflammatory chemokines in human macrophages and inhibited the overactivation of type 2 immunity in a mouse model of eosinophilic rhinosinusitis. Our findings advance the understanding of the heterogeneous immune microenvironment and the pathogenesis of CRS subtypes and identify potential therapeutic approaches for the treatment of CRS and potentially other type 2 immunity-mediated diseases.

Profile of Tissue Immunoglobulin E in Eosinophilic Chronic Rhinosinusitis with Nasal Polyps.

INTRODUCTION: Eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) exhibits a poorer prognosis than noneCRSwNP. The aim of this study was to analyze the potential of total immunoglobulin E (tIgE) and specific IgE (sIgE) levels in tissues for distinguishing and assessing eCRSwNP. METHODS: We enrolled 10 control and 88 CRSwNP patients. The clinical data of patients were collected before surgery. Nasal mucosa tissues were taken during surgery for measurements of tIgE, sIgE (weed pollen, epidermal and animal protein, mold, house dust, tree pollen), and subepithelial eosinophil (EOS) counts. The predictive significance of the potential predictors for eCRSwNP was assessed with receiver operating characteristic (ROC) curves. RESULTS: Nasal polyps tIgE and mold-sIgE were positively correlated with blood and tissue EOSs, comorbid allergic rhinitis and asthma, ethmoid score/total maxillary score ratio, visual analog scale, and CT score. The ROC curve analysis showed that tissue tIgE (p = 0.0004), mold-sIgE (p = 0.0030), blood EOS percentage (p = 0.0003), and absolute blood EOS count (p = 0.0010) acted as predictive factors for eCRSwNP. According to the cutoff value of tissue tIgE of 34.55 ku/L, patients with a high level were more likely to suffer from asthma (p = 0.016) and showed a significantly higher EOS count (p = 0.022), EOS percentage (p = 0.029), and tIgE (p = 0.002) in blood. CONCLUSION: Tissue tIgE and mold-sIgE had a significant relationship with the clinical and pathological characteristics of CRSwNP patients and might be reliable for distinguishing and assessing eCRSwNP.

The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1.

Abnormal accumulation of triglycerides in the liver, caused in part by increased de novo lipogenesis, results in non-alcoholic fatty liver disease and insulin resistance. Sterol regulatory element-binding protein 1 (SREBP1), an important transcriptional regulator of lipogenesis, is synthesized as an inactive precursor that binds to the endoplasmic reticulum (ER). In response to insulin signalling, SREBP1 is transported from the ER to the Golgi in a COPII-dependent manner, processed by proteases in the Golgi, and then shuttled to the nucleus to induce lipogenic gene expression; however, the mechanisms underlying enhanced SREBP1 activity in insulin-resistant obesity and diabetes remain unclear. Here we show in mice that CREB regulated transcription coactivator 2 (CRTC2) functions as a mediator of mTOR signalling to modulate COPII-dependent SREBP1 processing. CRTC2 competes with Sec23A, a subunit of the COPII complex, to interact with Sec31A, another COPII subunit, thus disrupting SREBP1 transport. During feeding, mTOR phosphorylates CRTC2 and attenuates its inhibitory effect on COPII-dependent SREBP1 maturation. As hepatic overexpression of an mTOR-defective CRTC2 mutant in obese mice improved the lipogenic program and insulin sensitivity, these results demonstrate how the transcriptional coactivator CRTC2 regulates mTOR-mediated lipid homeostasis in the fed state and in obesity.

Cross-talk between TH2 and TH17 pathways in patients with chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous disease with a spectrum of endotypes. TH2- and TH17-related cytokines are 2 central regulators involved in the inflammation associated with CRSwNP. OBJECTIVE: We sought to investigate the interregulation of TH2 and TH17 pathways in Chinese patients with CRSwNP. METHODS: Levels of key TH2- and TH17-related factors were measured in homogenates of polyp tissue obtained from patients with CRSwNP. The relationship of these factors and their expression in groups classified according to tissue IL-5 and IL-17 concentrations were analyzed. Cross-regulation of TH2 and TH17 cytokines and the effects of dexamethasone treatment were studied in dispersed nasal polyp cells. Associations between TH2- and TH17 related factors and comorbid atopic status and asthma, disease recurrence, and edema scores were also explored. RESULTS: Four CRSwNP groups were classified based on expression or nonexpression of mutually exclusive TH2- and TH17-related factors. The TH2 cytokines IL-4 and IL-13 inhibited expression of TH17-related factors, whereas the TH17 cytokines IL-17 and TGF-ß1 enhanced expression of TH2-related factors. Dexamethasone treatment inhibited both the TH2 and TH17 pathways. A patient's atopic status was related to their TH2 immune response. Edema scores were positively correlated with the TH2 pathway and negatively correlated with the TH17 pathway. CONCLUSION: The TH2 and TH17 pathways are mutually exclusive and regulate each other, favoring the development of a TH2 immune response in Chinese patients with CRSwNP.

Identification of glutathione S-transferase genes in Leptinotarsa decemlineata and their expression patterns under stress of three insecticides.

Glutathione S-transferases (GSTs) is a family of multifunctional enzymes that are involved in detoxification of poisonous compounds. In the present paper, the Leptinotarsa decemlineata genome and transcriptome dataset were mined and 30 GST genes were identified. These GSTs belonged to cytosolic (29 genes) and microsomal (1 gene) classes. Among them 3 GSTs (LdGSTe2, LdGSTs4, and LdGSTo3) possessed splice variants. Of the 29 cytosolic LdGSTs, 3, 10, 5, 4, 4, and 1 members were classified as delta, epsilon, omega, sigma, theta, and zeta subclasses respectively, along with 2 unclassified genes. Phylogenetic analysis suggest that epsilon, omega and sigma subclasses appear to undergo species-specific bloom. Moreover, most epsilon, omega and sigma GSTs are tandemly arranged in three chromosome scaffolds. To find GST candidates involving in insecticide detoxification, we tested the mRNA levels of 20 GST transcripts under stress of cyhalothrin, fipronil or endosulfan. Out of them, LdGSTe2a, LdGSTe2b, LdGSTo5 and LdGSTt1 were significantly overexpressed after exposure to each of the three insecticides. Two other genes were respectively upregulated after cyhalothrin (LdGSTe10 and LdGSTu2) or endosulfan (LdGSTd1 and LdGSTu2) treatment. The diversified expression responses to insecticide exposure suggest that the LdGSTs may depend on a functionally complex system to detoxify different classes of insecticides. In addition, our findings provide a base for a better understanding of the evolution of insecticide resistance, and functional research on specific GST genes.

Sarcomatoid carcinoma in the sinonasal cavity: A retrospective case series from a single institution.

OBJECTIVES: Sarcomatoid carcinoma (SC) is rare in the sinonasal cavity with poor prognosis. This study aimed to summarize the clinical characteristics, treatment and prognosis of patients with sinonasal SC from a single tertiary medical center. METHODS: Retrospective review of the clinical data of seven patients diagnosed with sinonasal SC from May 1988 to February 2021. RESULTS: Seven patients with SC in nasal cavity and paranasal sinus, including six males and one female, aged 34-77 years (mean, 58.3 years), were included in this case series. The common presenting symptoms included epistaxis and nasal obstruction. The tumor usually occurred in nasal cavity, maxillary sinus and ethmoid sinus. Most patients were diagnosed at advanced stages with the involvement of orbit and skull base. Open and endoscopic surgery were performed in five and two patients, respectively. The follow-up period ranged from 3 to 26 months. Four patients died of disease within one year, though two received adjuvant chemotherapy and one received radiotherapy. Two patients survived without recurrence at the end of follow-up, both of them underwent adjuvant radiotherapy. One patient received palliative re-irradiation due to local recurrence of the tumor three months after the surgery, however, this patient was lost to follow-up. CONCLUSIONS: Sinonasal SC is a rare entity with an aggressive behavior and poor prognosis. Early diagnosis and multimodality treatment are recommended to improve the survival.

Transcriptomic and Lipidomic Profiles in Nasal Polyps of Glucocorticoid Responders and Non-Responders: Before and After Treatment.

Background: The pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) and mechanisms underlying different responses to systemic glucocorticoids (GC) remain unclear. The major aim of this study was to explore the transcriptomic and oxidative lipidomic signatures and the effects of GC in patients with different clinical responses. Methods: Nasal polyp biopsies were obtained before and after 14-day oral GC treatment from 16 patients with CRSwNP, and normal nasal mucosa specimens were collected from 12 control subjects. RNA sequencing and oxidative lipidomics were performed, and differential gene expression analysis was conducted in the Responder and Non-responder groups at baseline and after treatment. Results: In the Responder group, GC significantly improved clinical symptoms and reduced tissue eosinophil infiltration. Meanwhile, GC led to a pronounced transcriptomic reversion with robust suppression of inflammatory responses and abnormal metabolism of extracellular matrix, as well as restoration of cilia function. However, non-responders were mainly characterized by epithelial hyperplasia and keratinization, with much less transcriptomic improvement after GC treatment. Higher expression of type 2 inflammatory molecules (CCL13, IGHE, CCL18, CCL23, CCR3, and CLC) with lower levels of LACRT, PPDPFL, DES, C6, MUC5B, and SCGB3A1 were related to a stronger clinical response to GC. Besides decreased prostaglandins and increased leukotrienes, increased dysregulation in other oxylipid mediators derived from polyunsaturated fatty acids was determined in nasal polyps, which was ameliorated by GC treatment. Conclusion: Systemic GC exert anti-inflammatory effects, improve tissue remodeling, restore cilia function, and ameliorate dysregulation of oxylipid mediator pathway in CRSwNP. GC-responders exhibited different transcriptomic signatures from non-responders.

The tight junction protein TJP1 regulates the feeding-modulated hepatic circadian clock.

Circadian clocks in the suprachiasmatic nucleus and peripheral tissues orchestrate behavioral and physiological activities of mammals in response to environmental cues. In the liver, the circadian clock is also modulated by feeding. However, the molecular mechanisms involved are unclear. Here, we show that TJP1 (tight junction protein 1) functions as a mediator of mTOR (mechanistic target of rapamycin) to modulate the hepatic circadian clock. TJP1 interacts with PER1 (period circadian regulator 1) and prevents its nuclear translocation. During feeding, mTOR phosphorylates TJP1 and attenuates its association with PER1, thereby enhancing nuclear shuttling of PER1 to dampen circadian oscillation. Therefore, our results provide a previously uncharacterized mechanistic insight into how feeding modulates the hepatic circadian clock.

PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation.

A change in the metabolic flux of glucose from mitochondrial oxidative phosphorylation (OXPHOS) to aerobic glycolysis is regarded as one hallmark of cancer. However, the mechanisms underlying the metabolic switch between aerobic glycolysis and OXPHOS are unclear. Here we show that the M2 isoform of pyruvate kinase (PKM2), one of the rate-limiting enzymes in glycolysis, interacts with mitofusin 2 (MFN2), a key regulator of mitochondrial fusion, to promote mitochondrial fusion and OXPHOS, and attenuate glycolysis. mTOR increases the PKM2:MFN2 interaction by phosphorylating MFN2 and thereby modulates the effect of PKM2:MFN2 on glycolysis, mitochondrial fusion and OXPHOS. Thus, an mTOR-MFN2-PKM2 signaling axis couples glycolysis and OXPHOS to modulate cancer cell growth.

OLFR734 Mediates Glucose Metabolism as a Receptor of Asprosin.

Asprosin is a fasting-induced hormone that promotes glucose production in the liver and stimulates appetite in the hypothalamus by activating the cAMP signaling pathway via an unknown G protein-coupled receptor (GPCR). However, the bona fide receptor of Asprosin is unclear. Here, we have identified that the olfactory receptor OLFR734 acts as a receptor of Asprosin to modulate hepatic glucose production. Olfr734 knockout mice show a blunted response to Asprosin, including attenuated cAMP levels and hepatic glucose production, and improved insulin sensitivity. As Olfr734 deficiency dramatically attenuates both fasting and high-fat-diet-induced glucose production, our results demonstrate a critical role of OLFR734 as a receptor of Asprosin to maintain glucose homeostasis during fasting and in obesity.

Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2.

The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events, such as the activation of viral glycoproteins and bacterial exotoxins. Small, non-toxic and highly active, furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria. In this study, a series of peptide inhibitors were designed and synthesized based on the C-terminal fragment of histone H1.2, which has an inhibitory effect on furin. Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity greatly. The most potent inhibitor, I4, with 14 amino acid residues has a Ki value of 17 nM for furin. Although most of the synthesized peptides were temporary inhibitors, the inhibitor I5, with nine amino acids, retained its full potency, even after a 3 h incubation period with furin at 37 degrees C. These inhibitors may potentially lead to the development of anti-viral and anti-bacterial drug compounds.

mTORC1 signaling in hepatic lipid metabolism.

The mechanistic target of rapamycin (mTOR) signaling pathway regulates many metabolic and physiological processes in different organs or tissues. Dysregulation of mTOR signaling has been implicated in many human diseases including obesity, diabetes, cancer, fatty liver diseases, and neuronal disorders. Here we review recent progress in understanding how mTORC1 (mTOR complex 1) signaling regulates lipid metabolism in the liver.

Fasting-induced hormonal regulation of lysosomal function.

Lysosomes are centers for nutrient sensing and recycling that allow mammals to adapt to starvation. Regulation of lysosome dynamics by internal nutrient signaling is well described, but the mechanisms by which external cues modulate lysosomal function are unclear. Here, we describe an essential role of the fasting-induced hormone fibroblast growth factor 21 (FGF21) in lysosome homeostasis in mice. Fgf21 deficiency impairs hepatic lysosomal function by blocking transcription factor EB (TFEB), a master regulator of lysosome biogenesis and autophagy. FGF21 induces mobilization of calcium from the endoplasmic reticulum, which activates the transcriptional repressor downstream regulatory element antagonist modulator (DREAM), and thereby inhibits expression of Mid1 (encoding the E3 ligase Midline-1). Protein phosphatase PP2A, a substrate of MID1, accumulates and dephosphorylates TFEB, thereby upregulating genes involved in lysosome biogenesis, autophagy and lipid metabolism. Thus, an FGF21-TFEB signaling axis links lysosome homeostasis with extracellular hormonal signaling to orchestrate lipid metabolism during fasting.

The potent inhibitory activity of histone H1.2 C-terminal fragments on furin.

Many physiologically important proproteins, pathogenic bacterial exotoxins and viral envelope glycoproteins are activated by the proprotein convertase furin, which makes furin inhibitor a hot target for basic research and drug design. Although synthetic and bioengineered inhibitors of furin have been well characterized, its endogenous inhibitor has not been directly purified from mammalian tissues to date. In this study, three inhibitors were purified from the porcine liver by using a combination of chromatographic techniques, and identified to be the C-terminal truncated fragments with different sizes of histone H1.2. The gene of porcine histone H1.2 was cloned and sequenced, further confirming the determined sequences. These three C-terminal fragments inhibited furin with Ki values around 2 x 10(-7) m while the full-length histone H1.2 inhibited it with a lesser activity, suggesting that the inhibitory activity relies on the C-terminal lysine-rich domain. Though the inhibition was temporary, these inhibitors were specific, and the reactive site of one C-terminal fragment was identified. A 36 amino acid peptide around the reactive site was synthesized, which could still inhibit furin with a Ki of 5.2 x 10(-7) m.

ß-Catenin-dependent lysosomal targeting of internalized tumor necrosis factor-α suppresses caspase-8 activation in apoptosis-resistant colon cancer cells.

The Wnt/ß-catenin pathway is constitutively activated in more than 90% of human colorectal cancer. Activated ß-catenin stimulates cell proliferation and survival, however, its antiapoptotic mechanisms are not fully understood. We show here that activated ß-catenin is required to suppress caspase-8 activation, but only in colon cancer cells that are resistant to tumor necrosis factor-α (TNF)-induced apoptosis. We found that lysosomal delivery of internalized TNF occurred at a faster pace in apoptosis-resistant than in apoptosis-sensitive colon cancer cells. Retardation of endosomal trafficking through vacuolar ATPase (V-ATPase) inhibition enhanced caspase-8 activation in apoptosis-resistant but not apoptosis-sensitive cells. Interestingly, knockdown of ß-catenin also prolonged TNF association with the early endosome and enhanced caspase-8 activation in apoptosis-resistant but not apoptosis-sensitive colon cancer cells. In a mouse model of inflammation-associated colon tumors, we found nuclear expression of ß-catenin, resistance to TNF-induced apoptosis, and reactivation of apoptosis in vivo after cotreatment of TNF with a V-ATPase inhibitor. Together these results suggest that activated ß-catenin can facilitate endosomal trafficking of internalized TNF to suppress caspase-8 activation in colon cancer cells.

Nuclear expression of ß-catenin promotes RB stability and resistance to TNF-induced apoptosis in colon cancer cells.

Tumor necrosis factor (TNF)-α promotes tumor development under chronic inflammation. Because TNF also activates caspase-8, selective inhibition of TNF-induced extrinsic apoptosis would be required for inflammation-associated tumor growth. In a mouse model of inflammation-associated colon carcinogenesis, we found nuclear expression of ß-catenin in tumors of wild-type, but not mutant, mice that were made resistant to TNF-induced apoptosis by a germline mutation blocking caspase cleavage of the retinoblastoma (RB) protein, despite similar frequencies of ß-catenin exon-3 mutations in these two genetic backgrounds. TNF-induced apoptosis was also attenuated in human colon cancer cell lines with genetically activated ß-catenin. However, we found that HCT116 cells, which contain an activated allele of ß-catenin but do not express nuclear ß-catenin, were sensitive to TNF-induced apoptosis. In HCT116 cells, TNF stimulated efficient RB cleavage that preceded chromatin condensation. In contrast, TNF did not induce RB cleavage in colon cancer cells expressing nuclear ß-catenin and these cells could be sensitized to basal and/or TNF-induced apoptosis by the knockdown of ß-catenin or RB. In the apoptosis-resistant colon cancer cells, knockdown of ß-catenin led to a reduction in the RB protein without affecting RB mRNA. Furthermore, ectopic expression of the caspase-resistant, but not the wild-type, RB re-established resistance to TNF-induced caspase activation in colon cancer cells without ß-catenin. Together, these results suggest that nuclear ß-catenin-dependent RB stabilization suppresses TNF-induced apoptosis in caspase-8-positive colon cancer cells.

Interaction of Mint3 with Furin regulates the localization of Furin in the trans-Golgi network.

Furin is a proprotein convertase that cycles between the plasma membrane, endosomes and the trans-Golgi network (TGN), maintaining a predominant distribution in the latter. Mint3, a member of the Mint protein family, is involved in the signaling and trafficking of membrane proteins. Until now, little has been known about the roles of Mint3 in the localization or trafficking of Furin. Here, using co-immunoprecipitation and immunofluorescence assays, we show that Mint3 interacts with Furin in the Golgi compartment of HeLa cells. Knockdown of endogenous Mint3 expression by RNA interference disrupts the TGN-specific localization of Furin and increases its distribution in endosomes. We further demonstrate that the phosphotyrosine-binding (PTB) domain of Mint3 is essential for the binding of Furin and that this binding affects the TGN-specific localization of Furin. Moreover, mutation studies of Furin indicate that Mint3 regulates Furin distribution mainly through interaction with the acidic peptide signal of Furin. Collectively, these data suggest that the interaction between the PTB domain of Mint3 and the acidic peptide signal of Furin regulates the specific localization of Furin in the TGN.

Possible role of histone H1 in the regulation of furin-dependent proprotein processing.

Histone H1 and its C-terminal lysine rich fragments were recently found to be potent inhibitors of furin, a mammalian proprotein convertase. However, its role in the regulation of furin-dependent proprotein processing remains unclear. Here we report that histone H1 efficiently blocks furin-dependent pro-von Willebrand factor (pro-vWF) processing in a dose-dependent manner. Coimmunoprecipitation and immunofluorescence studies confirmed that histone H1 could interact with furin, and the interaction mainly took place on the cell surface. We noted that histone H1 was released from cells undergoing necrosis and apoptosis induced by H(2)O(2). Our findings suggested that histone H1 might be involved in extracellular and/or intracellular furin regulation.