Richard C. Kurten

Enhanced degradation of EGF receptors by a sorting nexin, SNX1.

The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery. A protein, sorting nexin-1 (SNX1), was identified in a human cell line that bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code. SNX1 contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation. Thus, SNX1 is likely to play a role in sorting EGFR to lysosomes.

Mechanisms of Acetaminophen-Induced Hepatotoxicity: Role of Oxidative Stress and Mitochondrial Permeability Transition in Freshly Isolated Mouse Hepatocytes

Freshly isolated mouse hepatocytes were used to determine the role of mitochondrial permeability transition (MPT) in acetaminophen (APAP) toxicity. Incubation of APAP (1 mM) with hepatocytes resulted in cell death as indicated by increased alanine aminotransferase in the media and propidium iodide fluorescence. To separate metabolic events from later events in toxicity, hepatocytes were preincubated with APAP for 2 h followed by centrifugation of the cells and resuspension of the pellet to remove the drug and reincubating the cells in media alone. At 2 h, toxicity was not significantly different between control and APAP-incubated cells; however, preincubation with APAP followed by reincubation with media alone resulted in a marked increase in toxicity at 3 to 5 h that was not different from incubation with APAP for the entire time. Inclusion of cyclosporine A, trifluoperazine, dithiothreitol (DTT), or N-acetylcysteine (NAC) in the reincubation phase prevented hepatocyte toxicity. Dichlorofluorescein fluorescence increased during the reincubation phase, indicating increased oxidative stress. Tetramethylrhodamine methyl ester perchlorate fluorescence decreased during the reincubation phase indicating a loss of mitochondrial membrane potential. Inclusion of cyclosporine A, DTT, or NAC decreased oxidative stress and loss of mitochondrial membrane potential. Confocal microscopy studies with the dye calcein acetoxymethyl ester indicated that MPT had also occurred. These data are consistent with a hypothesis where APAP-induced cell death occurs by two phases, a metabolic phase and an oxidative phase. The metabolic phase occurs with GSH depletion and APAP-protein binding. The oxidative phase occurs with increased oxidative stress, loss of mitochondrial membrane potential, MPT, and toxicity.

TAS2R activation promotes airway smooth muscle relaxation despite β 2-adrenergic receptor tachyphylaxis

Recently, bitter taste receptors (TAS2Rs) were found in the lung and act to relax airway smooth muscle (ASM) via intracellular Ca(2+) concentration signaling generated from restricted phospholipase C activation. As potential therapy, TAS2R agonists could be add-on treatment when patients fail to achieve adequate bronchodilation with chronic β-agonists. The β(2)-adrenergic receptor (β(2)AR) of ASM undergoes extensive functional desensitization. It remains unknown whether this desensitization affects TAS2R function, by cross talk at the receptors or distal common components in the relaxation machinery. We studied intracellular signaling and cell mechanics using isolated human ASM, mouse tracheal responses, and human bronchial responses to characterize TAS2R relaxation in the context of β(2)AR desensitization. In isolated human ASM, magnetic twisting cytometry revealed >90% loss of isoproterenol-promoted decrease in cell stiffness after 18-h exposure to albuterol. Under these same conditions of β(2)AR desensitization, the TAS2R agonist chloroquine relaxation response was unaffected. TAS2R-mediated stimulation of intracellular Ca(2+) concentration in human ASM was unaltered by albuterol pretreatment, in contrast to cAMP signaling, which was desensitized by >90%. In mouse trachea, β(2)AR desensitization by β-agonist amounted to 92 ± 6.0% (P < 0.001), while, under these same conditions, TAS2R desensitization was not significant (11 ± 3.5%). In human lung slices, chronic β-agonist exposure culminated in 64 ± 5.7% (P < 0.001) desensitization of β(2)AR-mediated dilation of carbachol-constricted airways that was reversed by chloroquine. We conclude that there is no evidence for physiologically relevant cross-desensitization of TAS2R-mediated ASM relaxation from chronic β-agonist treatment. These findings portend a favorable therapeutic profile for TAS2R agonists for the treatment of bronchospasm in asthma or chronic obstructive lung disease.

Group 2 innate lymphocytes (ILC2) are enriched in active eosinophilic esophagitis

Author(s): Doherty, Taylor A; Baum, Rachel; Newbury, Robert O; Yang, Tom; Dohil, Ranjan; Aquino, Melissa; Doshi, Ashmi; Walford, Hannah H; Kurten, Richard C; Broide, David H; Aceves, Seema

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell lambda gt11 cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.

Coxiella burnetii Type IV Secretion-Dependent Recruitment of Macrophage Autophagosomes

ABSTRACT Coxiella burnetii is an intracellular Gram-negative bacterium that causes human Q fever, a flu-like disease that can progress to chronic, life-threatening endocarditis. In humans, C. burnetii infects alveolar macrophages and promotes phagosomal fusion with autophagosomes and lysosomes, establishing a unique parasitophorous vacuole (PV) in which to replicate. The pathogen uses a Dot/Icm type IV secretion system (T4SS) to deliver effector proteins to the host cytoplasm, where they alter cellular processes to benefit the pathogen. The T4SS is required for PV expansion and prevention of apoptosis, but little else is known about the role of the system during intracellular growth. Recent reports suggest that C. burnetii actively recruits autophagosomes to the PV to deliver nutrients to the pathogen and provide membrane for the expanding vacuole. In this study, we examined the role of the T4SS in mediating PV interactions with autophagosomes. We found that the autophagy-related proteins LC3 and p62 localized to wild-type PV but not to T4SS mutant organism-containing phagosomes in human macrophage-like cells, primary human alveolar macrophages, and Chinese hamster ovary cells. However, while lipidated LC3 levels were elevated regardless of T4SS activity, no p62 turnover was observed during C. burnetii growth in macrophages, suggesting that the pathogen recruits preformed autophagosomes. When the T4SS was activated 24 h after infection, autophagosome recruitment ensued, indicating that autophagosome interactions are dispensable for initial PV maturation to a phagolysosome-like compartment but are involved in vacuole expansion. Together, these results demonstrate that C. burnetii actively directs PV-autophagosome interactions by using the Dot/Icm T4SS.

Virulent Coxiella burnetii Pathotypes Productively Infect Primary Human Alveolar Macrophages

The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C. burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome‐like parasitophorous vacuole (PV). In vitro, C. burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogens infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C. burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C. burnetii–host cell interactions. C. burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non‐fused vacuoles, and lipid droplets were present in avirulent C. burnetii PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C. burnetii secretes effector proteins that control macrophage functions. Avirulent C. burnetii promoted sustained activation of Akt and Erk1/2 pro‐survival kinases and short‐termphosphorylation of stress‐related p38. Avirulent organisms also triggered a robust, early pro‐inflammatory response characterized by increased secretion of TNF‐α and IL‐6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro‐ and mature IL‐1β occurred in hAMs infected with avirulent C. burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN‐γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C. burnetii. Collectively, the current results demonstrate the hAM model is a human disease‐relevant platform for defining novel innate immune responses to C. burnetii.

Formoterol and salmeterol induce a similar degree of β2‐adrenoceptor tolerance in human small airways but via different mechanisms

Steroids prevent and reverse salbutamol‐induced β2‐adrenoceptor tolerance in human small airways. This study examines the effects of the long‐acting β2 agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization.

GSDMB induces an asthma phenotype characterized by increased airway responsiveness and remodeling without lung inflammation

Significance Because the SNP linking chromosome 17q21 to asthma is associated with increased gasdermin B (GSDMB) expression, we generated transgenic mice expressing increased levels of the human GSDMB transgene (hGSDMBZp3-Cre), which develop an asthma phenotype characterized by a spontaneous increase in airway responsiveness and airway remodeling (increased peribronchial smooth muscle) in the absence of the development of airway inflammation. These results challenge the current paradigm in asthma that airway inflammation induces smooth muscle remodeling and airway responsiveness, as these hGSDMBZp3-Cre mice develop increased airway-hyperresponsiveness and smooth muscle in the absence of airway inflammation. Furthermore, this study adds to our understanding of gene networks in asthma that we have identified can act in sequential pathways (i.e., GSDMB induces 5-lipoxygenase to induce TGF-β1). Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMBZp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.

Human Recombinant Vascular Endothelial Growth Factor Reduces Necrosis and Enhances Hepatocyte Regeneration in a Mouse Model of Acetaminophen Toxicity

We reported previously that vascular endothelial growth factor (VEGF) was increased in acetaminophen (APAP) toxicity in mice and treatment with a VEGF receptor inhibitor reduced hepatocyte regeneration. The effect of human recombinant VEGF (hrVEGF) on APAP toxicity in the mouse was examined. In early toxicity studies, B6C3F1 mice received hrVEGF (50 μg s.c.) or vehicle 30 min before receiving APAP (200 mg/kg i.p.) and were sacrificed at 2, 4, and 8 h. Toxicity was comparable at 2 and 4 h, but reduced in the APAP/hrVEGF mice at 8 h (p < 0.05) compared with the APAP/vehicle mice. Hepatic glutathione (GSH) and APAP protein adduct levels were comparable between the two groups of mice, with the exception that GSH was higher at 8 h in the hrVEGF-treated mice. Subsequently, mice received two doses (before and 10 h) or three doses (before and 10 and 24 h) of hrVEGF; alanine aminotransferase values and necrosis were reduced at 24 and 36 h, respectively, in the APAP/hrVEGF mice (p < 0.05) compared with the APAP/vehicle mice. Proliferating cell nuclear antigen expression was enhanced, and interleukin-6 expression was reduced in the mice that received hrVEGF (p < 0.05) compared with the APAP/vehicle mice. In addition, treatment with hrVEGF lowered plasma hyaluronic acid levels and neutrophil counts at 36 h. Cumulatively, the data show that treatment with hrVEGF reduced toxicity and increased hepatocyte regeneration in APAP toxicity in the mouse. Attenuation of sinusoidal cell endothelial dysfunction and changes in neutrophil dynamics may be operant mechanisms in the hepatoprotection mediated by hrVEGF in APAP toxicity.