Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-GMP signaling.

Bitter taste receptors (T2Rs) are GPCRs involved in detection of bitter compounds by type 2 taste cells of the tongue, but are also expressed in other tissues throughout the body, including the airways, gastrointestinal tract, and brain. These T2Rs can be activated by several bacterial products and regulate innate immune responses in several cell types. Expression of T2Rs has been demonstrated in immune cells like neutrophils; however, the molecular details of their signaling are unknown. We examined mechanisms of T2R signaling in primary human monocyte-derived unprimed (M0) macrophages (M[Formula: see text]s) using live cell imaging techniques. Known bitter compounds and bacterial T2R agonists activated low-level calcium signals through a pertussis toxin (PTX)-sensitive, phospholipase C-dependent, and inositol trisphosphate receptor-dependent calcium release pathway. These calcium signals activated low-level nitric oxide (NO) production via endothelial and neuronal NO synthase (NOS) isoforms. NO production increased cellular cGMP and enhanced acute phagocytosis ~ threefold over 30-60 min via protein kinase G. In parallel with calcium elevation, T2R activation lowered cAMP, also through a PTX-sensitive pathway. The cAMP decrease also contributed to enhanced phagocytosis. Moreover, a co-culture model with airway epithelial cells demonstrated that NO produced by epithelial cells can also acutely enhance M[Formula: see text] phagocytosis. Together, these data define M[Formula: see text] T2R signal transduction and support an immune recognition role for T2Rs in M[Formula: see text] cell physiology.

Small-molecule Akt-activation in airway cells induces NO production and reduces IL-8 transcription through Nrf-2.

BACKGROUND: The non-cancerous functions of Akt in the airway are understudied. In some tissues, Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) to produce nitric oxide (NO) that has anti-inflammatory effects. NO production has antibacterial and antiviral effects in the airway, and increasing NO may be a useful anti-pathogen strategy. Akt also stimulates the nuclear factor erythroid 2-related factor 2 (Nrf-2) transcription factor, which transcribes antioxidant genes. Therefore, we hypothesized that activation of the Akt/eNOS pathway, which also activates Nrf-2, may have protective effects in human airway cells against injury. METHODS: To directly test the effects of Akt signaling in the airway, we treated A549 and 16HBE cells as well as primary bronchial, nasal, and type II alveolar epithelial cells with small molecule Akt activator SC79. We examined the effects of SC79 on eNOS activation, NO production, Nrf-2 target levels, and interleukin-8 (IL-8) transcription during exposure to TNF-α or Pseudomonas flagellin (TLR5 agonist). Additionally, air-liquid interface bronchial cultures were treated with cadmium, an oxidative stressor that causes airway barrier breakdown. RESULTS: SC79 induced a ~ twofold induction of p-eNOS and Nrf-2 protein levels blocked by PI3K inhibitor LY294002. Live cell imaging revealed SC79 increased acute NO production. Quantitative RT-PCR showed a ~ twofold increase in Nrf-2 target gene transcription. TNF-α or flagellin-induced IL-8 levels were also significantly reduced with SC79 treatment. Moreover, the transepithelial electrical resistance decrease observed with cadmium was ameliorated by SC79, likely by an acute increase in tight junction protein ZO-1 levels. CONCLUSIONS: Together, the data presented here demonstrate SC79 activation of Akt induces potentially anti-pathogenic NO production, antioxidant gene transcription, reduces IL-8 transcription, and may protect against oxidative barrier dysfunction in a wide range of airway epithelial cells.

Angiotensin-(1-7)/mas inhibits apoptosis in alveolar epithelial cells through upregulation of MAP kinase phosphatase-2.

Earlier work from this laboratory showed that autocrine generation of angiotensin II and c-Jun-NH2-terminal kinase phosphorylation (p-JNK) are both required events in alveolar epithelial cell (AEC) apoptosis. Although earlier data showed that angiotensin-(1-7) [ANG-(1-7)] protects against AEC apoptosis, the pathways by which ANG-(1-7)/mas activation prevent JNK phosphorylation and apoptosis are poorly understood. Therefore, in the current study, it was theorized that ANG-(1-7) activates a mitogen-activated protein kinase phosphatase (MKP) and thereby reduces JNK phosphorylation to inhibit apoptosis and promote cell survival. This hypothesis was evaluated in the human A549 and mouse MLE12 AEC lines and primary cultures of human AECs. Cells were transfected with small-interfering RNAs, antisense oligonucleotides, or inhibitors specific for MKP-2 or mas, and were then assayed for phospho-JNK, caspase-9, loss of mitochondrial membrane potential, and nuclear fragmentation. Silencing of MKP-2 significantly prevented the blockade of all apoptotic markers by ANG-(1-7). Knockdown or blockade of mas receptor by antisense oligonucleotides or by the receptor antagonist A779, respectively, caused significant decreases in MKP-2, and simultaneously increased the apoptotic markers of caspase-9 activation and nuclear fragmentation. These data show that the ANG-(1-7)/mas pathway constitutively prevents JNK phosphorylation and apoptosis of AECs by maintaining activation of the JNK-selective phosphatase MKP-2, and further demonstrate the critical role of the ANG-(1-7) receptor mas in AEC survival.

Hyperoxia downregulates angiotensin-converting enzyme-2 in human fetal lung fibroblasts.

BACKGROUND: Angiotensin (ANG) II is involved in experimental hyperoxia-induced lung fibrosis. Angiotensin-converting enzyme-2 (ACE-2) degrades ANG II and is thus protective, but is downregulated in adult human and experimental lung fibrosis. Hyperoxia is a known cause of chronic fibrotic lung disease in neonates, but the role of ACE-2 in neonatal lung fibrosis is unknown. We hypothesized that ACE-2 in human fetal lung cells might be downregulated by hyperoxic gas. METHODS: Fetal human lung fibroblast IMR90 cells were exposed to hyperoxic (95% O2/5% CO2) or normoxic (21% O2/5% CO2) gas in vitro. Cells and culture media were recovered separately for assays of ACE-2 enzymatic activity, mRNA, and immunoreactive protein. RESULTS: Hyperoxia decreased ACE-2 immunoreactive protein and enzyme activity in IMR90 cells (both P < 0.01), but did not change ACE-2 mRNA. ACE-2 protein was increased in the cell supernatant, suggesting protease-mediated ectodomain shedding. TAPI-2, an inhibitor of TNF-α-converting enzyme (TACE/ADAM17), prevented both the decrease in cellular ACE-2 and the increase in soluble ACE-2 (both P < 0.05). CONCLUSION: These data show that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas. They also suggest that hyperoxia decreases ACE-2 through a shedding mechanism mediated by ADAM17/TACE.

Abrogation of ER stress-induced apoptosis of alveolar epithelial cells by angiotensin 1-7.

Earlier work showed that apoptosis of alveolar epithelial cells (AECs) in response to endogenous or xenobiotic factors is regulated by autocrine generation of angiotensin (ANG) II and its counterregulatory peptide ANG1-7. Mutations in surfactant protein C (SP-C) induce endoplasmic reticulum (ER) stress and apoptosis in AECs and cause lung fibrosis. This study tested the hypothesis that ER stress-induced apoptosis of AECs might also be regulated by the autocrine ANGII/ANG1-7 system of AECs. ER stress was induced in A549 cells or primary cultures of human AECs with the proteasome inhibitor MG132 or the SP-C BRICHOS domain mutant G100S. ER stress activated the ANGII-generating enzyme cathepsin D and simultaneously decreased the ANGII-degrading enzyme ACE-2, which normally generates the antiapoptotic peptide ANG1-7. TAPI-2, an inhibitor of ADAM17/TACE, significantly reduced both the activation of cathepsin D and the loss of ACE-2. Apoptosis of AECs induced by ER stress was measured by assays of mitochondrial function, JNK activation, caspase activation, and nuclear fragmentation. Apoptosis induced by either MG132 or the SP-C BRICHOS mutant G100S was significantly inhibited by the ANG receptor blocker saralasin and was completely abrogated by ANG1-7. Inhibition by ANG1-7 was blocked by the specific mas antagonist A779. These data show that ER stress-induced apoptosis is mediated by the autocrine ANGII/ANG1-7 system in human AECs and demonstrate effective blockade of SP-C mutation-induced apoptosis by ANG1-7. They also suggest that therapeutic strategies aimed at administering ANG1-7 or stimulating ACE-2 may hold potential for the management of ER stress-induced fibrotic lung disorders.

A four-part guide to lung immunology: Invasion, inflammation, immunity, and intervention.

Lungs are important respiratory organs primarily involved in gas exchange. Lungs interact directly with the environment and their primary function is affected by several inflammatory responses caused by allergens, inflammatory mediators, and pathogens, eventually leading to disease. The immune architecture of the lung consists of an extensive network of innate immune cells, which induce adaptive immune responses based on the nature of the pathogen(s). The balance of immune responses is critical for maintaining immune homeostasis in the lung. Infection by pathogens and physical or genetic dysregulation of immune homeostasis result in inflammatory diseases. These responses culminate in the production of a plethora of cytokines such as TSLP, IL-9, IL-25, and IL-33, which have been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Shifting the balance of Th1, Th2, Th9, and Th17 responses have been the targets of therapeutic interventions in the treatment of these diseases. Here, we have briefly reviewed the innate and adaptive i3mmune responses in the lung. Genetic and environmental factors, and infection are the major causes of dysregulation of various functions of the lung. We have elaborated on the impact of inflammatory and infectious diseases, advances in therapies, and drug delivery devices on this critical organ. Finally, we have provided a comprehensive compilation of different inflammatory and infectious diseases of the lungs and commented on the pros and cons of different inhalation devices for the management of lung diseases. The review is intended to provide a summary of the immunology of the lung, with an emphasis on drug and device development.

T2R bitter taste receptors regulate apoptosis and may be associated with survival in head and neck squamous cell carcinoma.

Better management of head and neck squamous cell carcinomas (HNSCCs) requires a clearer understanding of tumor biology and disease risk. Bitter taste receptors (T2Rs) have been studied in several cancers, including thyroid, salivary, and GI, but their role in HNSCC has not been explored. We found that HNSCC patient samples and cell lines expressed functional T2Rs on both the cell and nuclear membranes. Bitter compounds, including bacterial metabolites, activated T2R-mediated nuclear Ca2+ responses leading to mitochondrial depolarization, caspase activation, and ultimately apoptosis. Buffering nuclear Ca2+ elevation blocked caspase activation. Furthermore, increased expression of T2Rs in HNSCCs from The Cancer Genome Atlas is associated with improved overall survival. This work suggests that T2Rs are potential biomarkers to predict outcomes and guide treatment selection, may be leveraged as therapeutic targets to stimulate tumor apoptosis, and may mediate tumor-microbiome crosstalk in HNSCC.

Targeting the phosphoinositide-3-kinase/protein kinase B pathway in airway innate immunity.

The airway innate immune system maintains the first line of defense against respiratory infections. The airway epithelium and associated immune cells protect the respiratory system from inhaled foreign organisms. These cells sense pathogens via activation of receptors like toll-like receptors and taste family 2 receptors (T2Rs) and respond by producing antimicrobials, inflammatory cytokines, and chemokines. Coordinated regulation of fluid secretion and ciliary beating facilitates clearance of pathogens via mucociliary transport. Airway cells also secrete antimicrobial peptides and radicals to directly kill microorganisms and inactivate viruses. The phosphoinositide-3-kinase/protein kinase B (Akt) kinase pathway regulates multiple cellular targets that modulate cell survival and proliferation. Akt also regulates proteins involved in innate immune pathways. Akt phosphorylates endothelial nitric oxide synthase (eNOS) enzymes expressed in airway epithelial cells. Activation of eNOS can have anti-inflammatory, anti-bacterial, and anti-viral roles. Moreover, Akt can increase the activity of the transcription factor nuclear factor erythroid 2 related factor-2 that protects cells from oxidative stress and may limit inflammation. In this review, we summarize the recent findings of non-cancerous functions of Akt signaling in airway innate host defense mechanisms, including an overview of several known downstream targets of Akt involved in innate immunity.

Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds.

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF-dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including significant reduction of melanoma metastasis and bleomycin- induced fibrosis. Although these compounds are efficacious in these disease models, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent cotranscription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. We also show with genetic approaches that pirin modulates MRTF- dependent luciferase reporter activity. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-ß- induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which co crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

Bleomycin-Induced Neonatal Lung Injury Requires the Autocrine Pulmonary Angiotensin System.

BACKGROUND: Previous work from this laboratory demonstrated that apoptosis is regulated by a local angiotensin (ANG) system in alveolar epithelial cells (AECs). Autocrine generation of angiotensin II (ANGII) in response to endogenous or xenobiotic inducers is required for apoptosis in adult rat AECs and in AEC-derived human lung carcinoma cell line A549. Therefore, we hypothesized that a similar mechanism might also be involved in bleomycin (Bleo)-induced murine neonatal lung injury. METHODS: To investigate the local production of angiotensinogen (AGT) and ANGII in neonatal lung injury, lung explants were obtained from C57/BL6 wild type neonatal mice and were treated with Bleo in the presence or absence of an angiotensin converting enzyme (ACE) inhibitor. AGT protein, ANGII levels and caspase-9 were then measured. RESULTS: Exposure to Bleo significantly induced AGT protein (p<0.02), extracellular ANGII levels (p< 0.005) and the active form of caspase-9 (p<0.05) in neonatal lung tissue. Further, Bleo inducetion of both AGT protein and of caspase-9 were prevented by the ACE inhibitor lisinopril. CONCLUSION: These data clearly demonstrate the synthesis of AGT and ANGII in the lungs of neonates in response to Bleo. Furthermore, they suggest that manipulation of the angiotensin system may hold therapeutic potential for neonatal lung injury.

Molecular and cellular mechanisms of the inhibitory effects of ACE-2/ANG1-7/Mas axis on lung injury.

An established body of recent literature has demonstrated potent inhibitory effects of the angiotensin converting enzyme-2 (ACE-2)/ANG1-7/ Mas axis on acute lung injury and lung fibrogenesis. One of the mechanisms of this inhibition is the enzymatic action of ACE-2 to degrade its main substrate angiotensin (ANG) II, thereby reducing the injurious and profibrotic activities of this octapeptide. Another, potentially more important mechanism is the production by ACE-2 of the heptapeptide ANG1-7, which inhibits the actions of ANGII through its own receptor Mas, the product of the oncogene of the same name. Very recent efforts to define the molecular and cellular mechanisms of ANG1-7/Mas action have revealed a number of similar, but mechanistically distinct, pathways by which ANG1-7 and Mas act on various lung cell types to inhibit lung injury and fibrosis. In this review we summarize the beneficial actions of the ANG1-7/Mas pathway, specifically on lung cells in non-neoplastic lung injury. We also review the currently known downstream signaling mechanisms of the ANG1-7/Mas pathway in various lung cell types known to be key in acute injury and fibrogenesis.