Associations between <i>FKBP51</i> levels and eosinophilic inflammation in steroid-naïve patients with asthma.

<p>Associations between <i>FKBP51</i> mRNA levels normalized to <i>β₂ microglobulin</i> mRNA levels and a) blood and b) sputum eosinophil proportions (n = 31 each) and c) exhaled nitric oxide levels (n = 30) in steroid-naïve patients with asthma.</p

Effects of antitussive drugs on cough in <i>Ttll1</i>^−/− mice.

<p>(A, B, D, F, H) Graphs displaying the pre- and post-treatment number of coughs. (C) Graph displaying the dose–response relationship of the increased number of coughs in <i>Ttll1</i>^−/− mice. (E, G) Graph displaying the number of coughs evoked by inhaled capsaicin in wild-type (WT) mice treated with control and capsazepine (CPZ) or HC-030031. (A) Codeine phosphate (10 mg/kg) or saline was administered by gavage. Codeine phosphate significantly decreased cough in the <i>Ttll1</i>^−/− mice. (B) The <i>Ttll1</i>^−/− mice were nebulized with salbutamol (5 mg/ml) or phosphate-buffered saline (PBS). Salbutamol did not decrease cough in the <i>Ttll1</i>^−/− mice. (C) Moguisteine (3, 10, and 30 mg/kg) or control [0.5% dimethylsulfoxide (DMSO)] was intraperitoneally administered. Administration of 10 and 30 mg/kg moguisteine significantly inhibited cough in the <i>Ttll1</i>^−/− mice. (D) The <i>Ttll1</i>^−/− mice were nebulized with CPZ (300 μM) or control (10% DMSO). CPZ did not decrease cough in the <i>Ttll1</i>^−/− mice. (E) After treatment with nebulized CPZ (300 μM) or control (10% DMSO), the WT mice were nebulized with capsaicin to evoke cough. Nebulization with 300 μM CPZ was sufficient to inhibit cough evoked by capsaicin in the WT mice. (F) Vehicle (0.5% methyl cellulose in sterile saline) or HC-030031 (300 mg/kg) was administered intraperitoneally to the <i>Ttll1</i>^−/− mice. HC-030031 did not decrease cough in the <i>Ttll1</i>^−/− mice. (G) After administration of HC-030031 (300 mg/kg) or vehicle, the WT mice were nebulized with acrolein (10 mM) to evoke cough. Administration of HC-030031 was sufficient to inhibit cough evoked by acrolein in the WT mice. (H) Lidocaine (4%) or saline was administered to each nostril. Lidocaine decreased cough in the <i>Ttll1</i>^−/− mice [bars indicate median values; n = 5–10 mice in each group; *P < 0.05, number of coughs compared between pre- and post-treatment (in A−D, F, H) or between control and treated groups (in E and G) using Wilcoxon signed-rank test; †P < 0.05, increased number (post-treatment–pretreatment) compared with that in the control group using Mann–Whitney U-test; NS = not significant].</p

Nasal mucociliary clearance was decreased in <i>Ttll1</i>^−/− mice.

<p>(A) Tosufloxacin (50 mg/kg) was administered to <i>Ttll1</i>^−/− mice with rhinosinusitis to inhibit cough for 7 days. Treatment with tosufloxacin did not decrease cough (bars indicate median values, n = 4 per group; NS = not significant). (B) Nasal sections (coronal) of the <i>Ttll1</i>^−/− mice treated with tosufloxacin. Neutrophils were decreased in the mucus. However, mucus accumulation persisted (*) (n = 4). (C) Representative computed tomography scans of the coronal nasal cavity. The area occupied by contrast material is bordered with a red line. (D) We calculated the percent changes in the area of contrast material [(area at 30 min − area at 150 min)/area at 30 min] to assess clearance of contrast material (mean ± SEM, n = 4, *P < 0.005 by two-tailed Student's t test). (E) Concentration of Evans blue in the nasal cavity and stomach 90 min after administration. In the <i>Ttll1</i>^−/− mice, a larger amount of Evans blue persisted in the nasal cavity and a lesser amount was swallowed compared with that in the wild-type (WT) mice (mean ± SEM, n = 5, *P < 0.005 by two-tailed Student's t test).</p

Respiratory reflexes in humans, guinea pigs, wild-type (WT) mice, and <i>Ttll1</i>^{<i>−/−</i>} mice.

<p>(A−D): Charts exhibiting airflow patterns of respiratory reflexes. Expiratory flow is indicated by the plus sign (upward) and inspiratory flow is indicated by the minus sign (downward). Airflow patterns of human reflexes (A). Airflow through the nose and mouth induced by cough, sneeze, and the expiration reflex was recorded using a spirometer (n = 3). Cough and the expiration reflex were evoked by inhaled capsaicin. Sneeze was evoked by mechanical stimuli applied by rubbing the nasal cavity with a tapered tissue paper. Airflow patterns of reflexes in guinea pigs (B) and WT mice (C; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s002" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s005" target="_blank">S4</a> Videos). Airflows of cough and sneeze were analyzed using a whole body plethysmograph (WBP). Cough was evoked by inhaled citric acid in guinea pigs and capsaicin in mice. Sneeze was induced by intranasal instillation of ovalbumin in sensitized animals. In (A–C), cough and sneeze showed one-peak and two-peak expiration patterns with preceding inspiration, respectively. Airflow patterns of reflexes in <i>Ttll1</i>^−/− mice (D; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s006" target="_blank">S5</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s008" target="_blank">S7</a> Videos). These reflexes were analyzed by WBP and classified into three patterns: (i) one-peak expiration with preceding inspiration, (ii) two-peak expiration with preceding inspiration, and (iii) one-peak expiration without preceding inspiration. Patterns (i) and (ii) corresponded to cough and sneeze patterns, respectively. #: expiration during eupneic breathing, which was not accompanied by characteristic sound and motion. (E) Representative photos recorded by videofluoroscopy. The <i>Ttll1</i>^−/− mice were placed in a WBP device. Inspiration and expiration phases in cough and normal breathing are shown. Solid lines indicate the diaphragm of the <i>Ttll1</i>^−/− mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141823#pone.0141823.s009" target="_blank">S8 Video</a>). (F) The bar graph shows the calculated amplitude of the diaphragm while coughing [= b–a in (E)] and normal breathing [= d–c in (E)]. The maximal distance [two-headed arrow in (E)] between the dotted line connecting the costophrenic angles and the diaphragm [solid line in (E)] as measured during inspiration and expiration while coughing and normal breathing. Diaphragm motion in the <i>Ttll1</i>^−/− mice was larger during coughing than during normal breathing (n = 3; mean ± SEM; *P = 0.006 by two-tailed Student's t-test). (G) Number of respiratory reflexes of the <i>Ttll1</i>^−/− mice in ten minutes (mean ± SEM, n = 10).</p

Representative images of immunostaining of purified blood eosinophils and non-eosinophils from healthy controls.

<p>Case 1 (46-year-old female), case 2 (36-year-old female), case 3 (35-year-old male). Column A: staining with anti-FKBP51 antibody, column B: merged image of staining with anti-major basic protein antibody (MBP) and anti-FKBP51 antibody. Red indicates MBP, and green indicates FKBP51.</p

<i>FKBP51</i> levels in induced sputum cells in patients with asthma.

<p><i>FKBP51</i> mRNA levels normalized to <i>β₂ microglobulin</i> mRNA levels in induced sputum cells became progressively higher from steroid-naïve asthmatic patients (naïve, n = 31), to mild to moderate asthmatic patients on inhaled corticosteroid (mild to moderate, n = 6), and then to severe persistent asthmatic patients on inhaled corticosteroid (severe, n = 22) (p<0.0001 by the Kruskal-Wallis test). *Significant by the Wilcoxon rank-sum test. Values and bars represent means.</p

Representative images of immunostaining of sputum cells from asthmatic patients.

<p>Case 1 (68-year-old male) and case 2 (72-year-old female) were steroid-naïve patients. Case 3 (79-year-old male) and case 4 (55-year-old female) were patients with severe persistent asthma on high-dose inhaled corticosteroid. Column A: staining with anti-FKBP51 antibody, column B: merged image of staining with anti-major basic protein antibody (MBP) and anti-FKBP51 antibody. Red indicates MBP, and green indicates FKBP51.</p

Patients’ characteristics.

<p>Values are given as means ± SD or medians (range).</p>*<p>included four patients with mild and two with moderate persistent asthma.</p>†<p>with the χ² test or analysis of variance.</p>‡<p>Patients were considered atopic when they were positive for one or more serum allergen-specific IgE antibodies against house dust, Japanese cedar pollen, mixed gramineae pollen, mixed weed pollen, mixed mold, cat dander, dog dander, and <i>Trichophyton rubrum</i>.</p>§<p>Equivalent to fluticasone propionate.</p>¶<p>by Kruskal Wallis test,</p>#<p>by unpaired t-test or analysis of variance after data were log-transformed.</p><p>Abbreviations: ICS, inhaled corticosteroid; FEV₁; forced expiratory volume in one second.</p

Laryngeal stimuli by postnasal drip-evoked cough in mice.

<p>(A) Contrast material in the nasal airway and upper airway was scanned by computed tomography (CT). CT shows optimal nasal airway obstruction with contrast material (arrows). (B) Physical blockade of the nasal airway to dam postnasal drip. Cyanoacrylate glue was placed in the nasal airway of <i>Ttll1</i>^−/− mice, as illustrated. Sagittal section of the nasal airway stained with hematoxylin and eosin showing obstruction of the nasal airway with cyanoacrylate glue (#). (C) Graph displaying the pre- and post-treatment number of coughs in the <i>Ttll1</i>^−/− mice. Cough in the <i>Ttll1</i>^−/− mice was completely inhibited by nasal airway blockade with cyanoacrylate glue (bars: median values, n = 7, *P = 0.02 by Wilcoxon signed-rank test). (D−F) Artificial postnasal drip in the wild-type (WT) mice (n = 5). A blue-colored polyvinyl alcohol (PVAL) solution was intranasally administered to the WT mice to mimic postnasal drip. The photos show the lower jaw (D) and lung (F), and the photomicrographs show sagittal sections of the larynx (E) after administration of the PVAL solution. The PVAL solution (blue) was found in the larynx (white and black arrowheads). Bar, 1 mm. There was no finding of aspiration of the PVAL solution in the trachea and lungs (E and F). (G) Graph showing the pre- and post-treatment number of coughs in the WT mice. A PVAL solution (artificial postnasal drip) was intranasally administered to the WT mice. Cough was evoked by an artificial postnasal drip in the WT mice (n = 4).</p

Increased cough sensitivity in <i>Ttll1</i>^−/− mice.

<p>Wild-type (WT) and <i>Ttll1</i>^−/− mice were nebulized with saline and capsaicin (10 and 50 μM). A graph displaying the increased number of coughs (post-treatment–pretreatment). Even low doses (10 μM) of capsaicin increased the number of coughs in the <i>Ttll1</i>^−/− mice but not in the WT mice. High doses (50 μM) of capsaicin increased the number of coughs in the WT and <i>Ttll1</i>^−/− mice. (n = 5 mice per group; mean ± SEM; *P < 0.05, the increased numbers of coughs were compared between the WT and <i>Ttll1</i>^−/− mice; †P < 0.05 and ‡P < 0.01, the increased numbers of coughs in comparison with those induced by saline; Mann–Whitney U-test).</p