Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma

<div><p>β₂-adrenergic receptor (β₂AR) agonists (β₂-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β₂ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β₂ARs as a measure of resensitization. Despite significant accumulation of β₂ARs in the endosomes of asthmatic HASMCs, endosomal β₂ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β₂AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β₂AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.</p></div

βAR function in primary human airway smooth muscle cells (HASMCs) from lungs of non-asthma (Non- asthmatic ASM) and asthma (Asthmatic ASM) patients.

<p><b>a</b>, Non- asthmatic ASM and asthmatic ASM cells were stimulated with β-agonist albuterol (0, 5, 10 & 20 minutes (min)). The cell were lysed and assessed for the ability to generate cAMP. *p<0.005 vs. non-asthmatic ASM or asthmatic ASM 0 min (untreated), #p<0.05 vs. asthmatic ASM 5, 10, 20 min, (n = 6 per group, 6 non-asthmatic ASM and 6 asthmatic ASM). <b>b</b>, Plasma membranes were isolated from non-asthmatic ASM and asthmatic ASM cells following pre-treatment of cells with albuterol for 0, 5, 10, and 20 min. The cell-free membranes were stimulated with albuterol to measure adenylyl cyclase activity by providing radioactive ^32[P]γ-ATP and measuring cAMP generation. *p<0.01 vs. respective <i>in vitro</i> vehicle stimulation, #p<0.05 vs. ASM <i>in vitro</i> albuterol stimulated 0 and 5 min, (n = 6/group). V, Vehicle; Alb, Albuterol (β-agonist).</p

Assessment of βAR desensitization in non-asthmatic ASM and asthmatic ASM cells.

<p><b>a</b>, To determine whether βAR are differentially phosphorylated in non-asthmatic ASM and asthmatic ASM cells, the cells were plated on cover slips and β₂AR phosphorylation was visualized by confocal microscopy using anti-phospho-S-355/356 β₂AR antibody (green) (Scale-100 μM) (n = 5/group). <b>b</b>, Plasma membranes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to ^[125]I-CYP (cyanopindalol) βAR binding at saturation concentration of 250 pmol. *p<0.005 vs. non-asthmatic ASM, (n = 7/group). <b>c</b>, To investigate changes in desensitization βAR components lysates (100 μg) from non-asthmatic ASM and asthmatic ASM cells were immunoblotted for ubiquitously expressed GRKS, GRK2, 3, 5 or 6. The blots were stripped for each probing. Furthermore, the blots were immunoblotted for β-arrestin 1 and 2. Actin was blotted as loading control (n = 7/group).</p

Measure of βAR resensitization in non-asthmatic ASM and asthmatic ASM cells (βAR function, density and distribution).

<p><b>a</b>, To obtain a measure of resensitization that mediates βAR dephosphorylation leading to generation of agonist ready receptors in the endosomes, endosomes were isolated from non-asthmatic ASM and asthmatic ASM cells, and subjected to various assays. <b>b</b>, Plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to ^[125]I-CYP (cyanopindalol) βAR binding at saturation concentration of 250 pmol. *p<0.005 vs. Non-ASM, (n = 4/group). <b>c</b>, Plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to cell-free membrane associated β-agonist (isoproterenol, ISO)-stimulated adenylyl cyclase activity. *p<0.01 vs. respective cell-free vehicle (V) stimulation, #p<0.05 vs. asthmatic ASM ISO, (n = 4/group). <b>d</b>, Upper panel, plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells (100 μg) were immunoblotted using anti-phospho-S-355/356 β₂AR antibody. Lower panel, densitometry for phospho- β₂AR (n = 4/group). *p<0.01 vs. plasma membrane (P) non-asthmatic ASM, #p<0.05 vs. endosomes (E) non-asthmatic ASM. V, Vehicle, ISO, isoproterenol (β-agonist), P, Plasma membrane, E, Endosome.</p

Measure of βAR resensitization in Non-ASM and ASM HASMCs (measure of resensitization components PI3Kγ and PP2A activity).

<p><b>a</b>, Upper panel, PI3Kγ was immunoprecipitated from plasma membrane and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells (120 μg) and the washed immunoprecipitates were subjected to <i>in vitro</i> lipid kinase assays. Lower panel, densitometry for PI3Kγ (n = 4/group). *p<0.01 vs. plasma membrane (P) non-asthmatic ASM, #p<0.0001 vs. endosomes (E) non-asthmatic ASM. <b>b</b>, PP2A was immunoprecipitated from (100 μg) of non-asthmatic ASM and asthmatic ASM cells using anti-PP2A antibodies and the immunoprecipitates were subjected to <i>in vitro</i> phosphatase assay with malachite green as a read for activity, (n = 4/group). *p<0.001 vs. non-asthmatic ASM,). <b>c</b>, Illustration depicting the loss of resensitization as a underlying cause for βAR dysfunction leading to paradoxical loss in β-agonist response. Left Panel (non-asthmatic ASM conditions), βAR activation by β-agonist leads to desensitization by phosphorylation and resensitization by dephosphorylation via PP2A resulting in normal βARs recycling. Right Panel (asthmatic ASM conditions), desensitization is well understood but, little is known of resensitization mechanisms. Since we have shown that βAR resensitization is regulated by PI3Kγ-PP2A axis, assessment in the endosomes shows marked increase in PI3Kγ activity associated with loss in PP2A activity. Based on this observation, we propose that asthma HASMCs are characterized by loss in βAR resensitization wherein, increased PI3Kγ inhibits PP2A activity blocking receptor resensitization resulting in accumulation of βARs in the endosomes.</p

Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans

<div><p>Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a <i>paramyxovirus</i> infection in humans.</p></div

RSV iDVGs stimulate an IRF1/IFNL1-mediated antiviral response.

<p>A549 cells were infected with RSV-LD or RSV-HD at a moi of 1.5 TCID₅₀/cell. Expression of (A) RSV G and antiviral genes mRNA, and (B) protein level of IFNB1 and IFNL1/3 in the cultures supernatants at 24 h post infection (*p<0.05 by one way unpaired t-test). (C, D) Cells were lysed or fixed at 2, 6, 12, and 24 h post infection for western blot (WB) and IFA. (C) For WB, nuclear and cytosolic fractions were immunoblotted for IRF1, GAPDH, and Histone 3. (D) For IFA, cells were co-stained for IRF1 (green, left panel), RSV F + G proteins (red in merged panel), and nuclei (blue). (E) Quantification of nuclear IRF1 upon iDVGs stimulation at designated time points post RSV-HD infection based on IFA images. (F, G) D54 control cells and D54 cells overexpressing IRF1 (D54-IRF1) were infected with RSV-HD at a moi of 1.5 TCID₅₀/cell for 6 h. (F) IRF1 and IRF3 protein detected by WB from whole cell lysates. (G) Expression of RSV G and IFNL1 mRNA. (H, I) A549 cells were mock transfected (WT) or transfected with control siRNA (si-C), or IRF1 siRNA (si-1). After 40 h, the cells were mock infected or infected with RSV-HD at moi of 1.5 TCID₅₀/cell. (H) WB for IRF3 and IRF1 was performed to confirm specific knockdown of IRF1 protein. (I) Expression of RSV G and other antiviral genes at 10 h post infection. Gene expression is shown as copy number relative to a house keeping gene expression index determined from the expression of ACTB (β-actin) and GAPDH. All error bars indicate mean ± SEM of at least three independent experiments (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test).</p

RSV DVGs prevent viral pathogenesis in vivo.

<p>(A) Hep2 cells were infected with RSV-LD or HD at a moi of 1.5 TCID₅₀/cell and DVGs were detected by PCR at the indicated times. Details of the PCR assay can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005122#ppat.1005122.s001" target="_blank">S1 Fig</a> and sequences of the amplicons labeled with a star can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005122#ppat.1005122.s002" target="_blank">S2 Fig</a> Base pair size references are indicated in the gel. (B) Mice weight loss was monitored overtime. Error bars indicate standard deviation of data pooled from two independent experiments (n = 7–9 mice per group total; **p<0.01, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test. Variance was not significantly different between groups as per Bartlett’s test). (C) Representative H&E staining for lung sections from mock, RSV-LD, or RSV-HD-infected mice on day 2 post infection. Picture magnification: 10X; insert is a digital amplification. Red arrows indicate alveolar cellular infiltrate. (D) Pathology score for alveolar infiltration in the lung (n = 6 mice per group; **p<0.01, by two-tailed Mann Whitney test). (E) Differential counts from cytospins from mice bronchoalveolar lavage (BAL) on day 2 post infection (Mono: monocytes and macrophages, PMNs: polymorphonuclear cells; ***p<0.001 by two-way ANOVA with Bonferroni’s post hoc test, n = 5–8 mice per group). (F) Representative cytospin images (20X). (G) Representative flow cytometry plots from whole lung single cell suspensions on day 2 post infection. Plots are pre-gated in singlets, live, CD45⁺CD11b⁺ cells. (H) Quantification of different cell types in the lung of infected mice on day 2 post infection (****p<0.0001 by two-way ANOVA with Bonferroni’s post hoc test, n = 5–8 mice per group, Alv. Macs: alveolar macrophages). (I) Expression of pro-inflammatory genes in whole lung tissue on day 2, 5, and 8 post infection. (n = 3–5 mice per group, *p<0.05, **p<0.01 by one-way ANOVA with Bonferroni’s post hoc test).</p

Host-intrinsic factors determine the response to iDVGs in the human lung.

<p>(A) Images of precision cut lung slices from human lungs (hPCLS) infected for 24 h with 10⁶ RSV-GFP TCID₅₀/slice. Top: Overlay of bright field and fluorescence channels; Bottom: fluorescence channel alone. (B) Gene expression from hPCLS infected with 10⁷ TCID₅₀/slice of RSV-LD or HD for up to 5 days (n = 7–8, grey numbers indicate individual lung donor). Results show paired data, numbers correspond to different donors. (*p<0.05, **p<0.01 by one-tailed Wilcoxon matched-pairs signed rank test). (C) Ratio of gene expression in RSV-HD and RSV-LD infected tissue from different donors (P3-P9). Ratio>1: HD induced a higher gene expression than LD. (D) PCR for DVGs in hPCLS infected with 10⁶ TCID₅₀/slice of RSV-LD. (E) Gene expression from (D). Error bars indicate mean ± SEM of three slices from the same patient.</p

iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.

<p>(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).</p