NLRP3 knockout in mice provided protection against Serratia marcescens-induced acute pneumonia by decreasing PD-L1 and PD-1 expression in macrophages.

Antibiotic-resistant Serratia marcescens (Sm) is known to cause bloodstream infections, pneumonia, etc. The nod-like receptor family, pyrin domain-containing 3 (NLRP3), has been implicated in various lung infections. Yet, its role in Sm-induced pneumonia was not well understood. In our study, we discovered that deletion of Nlrp3 in mice significantly improved Sm-induced survival rates, reduced bacterial loads in the lungs, bronchoalveolar lavage fluid (BALF), and bloodstream, and mitigated the severity of acute lung injury (ALI) compared to wild-type (WT) mice. Mechanistically, we observed that 24 h post-Sm infection, NLRP3 inflammasome activation occurred, leading to gasdermin D NH2-terminal (GSDMD-NT)-induced pyroptosis in macrophages and IL-1ß secretion. The NLRP3 or NLRP3 inflammasome influenced the expression PD-L1 and PD-1, as well as the count of PD-L1 or PD-1-expressing macrophages, alveolar macrophages, interstitial macrophages, PD-L1-expressing neutrophils, and the count of macrophage receptors with collagenous structure (MARCO)-expressing macrophages, particularly MARCO+ alveolar macrophages. The frequency of MARCO+ alveolar macrophages, PD-1 expression, particularly PD-1+ interstitial macrophages were negatively or positively correlated with the Sm load, respectively. Additionally, IL-1ß levels in BALF correlated with three features of acute lung injury: histologic score, protein concentration and neutrophil count in BALF. Consequently, our findings suggest that Nlrp3 deletion offers protection agaisnt acute Sm pneumonia in mice by inhibiting inflammasome activation and reducing Sm infection-induced PD-L1/PD-1 or MARCO expression, particularly in macrophages. This highlights potential therapeutic targets for Sm and other gram-negative bacteria-induced acute pneumonia.

IL-24 Contributes to Neutrophilic Asthma in an IL-17A-Dependent Manner and Is Suppressed by IL-37.

PURPOSE: Neutrophilic asthma is associated with asthma exacerbation, steroid insensitivity, and severe asthma. Interleukin (IL)-24 is overexpressed in asthma and is involved in the pathogenesis of several allergic inflammatory diseases. However, the role and specific mechanism of IL-24 in neutrophilic asthma are unclear. We aimed to elucidate the roles of IL-24 and IL-37 in neutrophilic asthma, the relationships with IL-17A and the mechanisms regulating neutrophilic asthma progression. METHODS: Purified human neutrophils were isolated from healthy volunteers, and a cell coculture system was used to evaluate the function of IL-24 in epithelium-derived IL-17A-dependent neutrophil migration. IL-37 or a small interfering RNA (siRNA) targeting IL-24 was delivered intranasally to verify the effect in a murine model of house dust mite (HDM)/lipopolysaccharide (LPS)-induced neutrophilic asthma. RESULTS: IL-24 enhanced IL-17A production in bronchial epithelial cells via the STAT3 and ERK1/2 signaling pathways; this effect was reversed by exogenous IL-37. Anti-IL-17A monoclonal antibodies reduced neutrophil chemotaxis induced by IL-24-treated epithelial cells in vitro. Increased IL-24 and IL-17A expression in the airway epithelium was observed in HDM/LPS-induced neutrophilic asthma. IL-37 administration or IL-24 silencing attenuated neutrophilic asthma, reducing IL-17A levels and decreasing neutrophil airway infiltration, airway hyperresponsiveness, and goblet cell metaplasia. Silencing IL-24 inhibited T-helper 17 (Th17) immune responses, but not Th1 or Th2 immune responses, in the lungs of a neutrophilic asthma model. CONCLUSIONS: IL-24 aggravated neutrophilic airway inflammation by increasing epithelium-derived IL-17A production, which could be suppressed by IL-37. Targeting the IL-24/IL-17A signaling axis is a potential strategy, and IL-37 is a potential candidate agent for alleviating neutrophilic airway inflammation in asthma.

Cough Inhibition Activity of Schisandra chinensis in Guinea Pigs.

Chronic cough is very common in respiratory clinics, and no effective drugs are available. Schisandra chinensis (Turcz.) Baill. (S. chinensis), an important traditional Chinese medicine, has been extensively prescribed for patients with a persistent cough. Preliminary research indicated that 95% ethanol extracts (EE) of S. chinensis showed remarkable antitussive activity in guinea pigs exposed to cigarette smoke (CS). To find out the antitussive ingredients of S. chinensis, EE was divided into four fractions according to the polarity: petroleum ether extract (PEE), ethyl acetate extract (ECE), n-butyl alcohol extract, and residue extract. The antitussive, antioxidant, and anti-inflammatory effects of the four fractions were evaluated in a guinea pig model of cough hypersensitivity induced by CS exposure. Eighteen main constituents of the two effective fractions, PEE and ECE, were identified using ultra-high-pressure liquid chromatography electronic spray ion time-of-flight mass spectrometry. The cough inhibition activities of compound 1, 3, 9, 10, 17 were evaluated on citric acid induced acute cough guinea pigs. The results showed that the antitussive activity of EE was almost all contained in PEE and ECE. The 16 major peaks in PEE were identified as 15 lignans (1-12 and 14-16) and 1 triterpene (compound 13), and 3 major peaks (1, 17, and 18) in ECE were also identified as lignans. Three doses of five compounds brought about a significant decrease in number of cough efforts (P < .01), and the cough inhibition rates were between 40.9% and 85.1%. Therefore, lignans are the antitussive ingredients of S. chinensis.

STAT1 participates in the induction of substance P expression in airway epithelial cells by respiratory syncytial virus.

PURPOSE: The regulation effect and mechanism of respiratory syncytial virus (RSV) infection on the expression of tachykinin substance P (SP) in airway epithelial cells was investigated. METHODS: The regulation of SP expression by RSV was investigated in the BEAS-2B airway epithelial cell line. RT-qPCR, immunofluorescence, and ELISA assay were used to examine the expression of the SP encoding gene TAC1, the intracellular SP protein expression, and the extracellular SP secretion. RESULTS: The mRNA expression of TAC1 and the intracellular SP protein level in BEAS-2B cells were significantly enhanced by RSV infection with multiplicity of infection (MOI) values of both 1 and 0.1 at 48 hours post infection. Heat-inactivated and UV-inactivated RSV, but not live RSV, significantly induced SP secretion in both control BEAS-2B cells and CX3CR1 receptor knockout cells without affecting the TAC1 gene expression or cell viability. RSV G protein (2-10 µg/ml) and fractalkine (10-50 ng/ml), both CX3CR1 receptor ligands, did not affect SP secretion in BEAS-2B cells. Inhibition of STAT1 phosphorylation by fludarabine (1 µM) markedly reduced the RSV-induced TAC1 gene expression and antagonized the inhibition of RSV replication by interferon-α in BEAS-2B cells. CONCLUSIONS: STAT1 participates in RSV infection-induced SP expression in airway epithelial cells.

Antitussive activity of the Schisandra chinensis fruit polysaccharide (SCFP-1) in guinea pigs models.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra chinensis (Turcz.) Baill. (S. chinensis), locally known as "Wuweizi", has been used in the treatment of chronic cough as prescription medications of Traditional Chinese Medicine for thousands of years. However, the components of antitussive activity of S. chinensis and the mechanism are poorly understood. AIM OF THE STUDY: This study aims to investigate the antitussive activity of polysaccharides extracted from S. chinensis. MATERIALS AND METHODS: S. chinensis fruit polysaccharide-1 (SCFP-1) was extracted by 95% ethanol and distilled water successively, and then the water extraction was isolated with chromatographic columns. The preliminary characterization of SCFP-1 was analyzed by gel permeation chromatography (GPC), gas chromatography-mass spectrometry (GC-MS) and some other recognized chemical methods. Antitussive potential of SCFP-1 was estimated at dose of 250, 500, and 1000mg/kg respectively by peroral administration in a guinea pigs model with cough hypersensitivity induced by cigarette smoke (Chronic cough model) or acute cough guinea model induced by citric acid (Acute cough model). Also, the time-dependent antitussive effect of SCFP-1 were evaluated with acute cough model, and compared with codeine. RESULTS: The molecular of SCFP-1 was 3.18×104Da, mainly being composed of glucose and arabinose (66.5% and 29.4%, respectively). Peroral administration of SCFP-1 at 250, 500, and 1000mg/kg showed remarkable suppressive effects respectively on cough in both of chronic cough model and acute cough model. Meanwhile, inflammatory cell in BALF and some typical characteristics of nonspecific airway inflammation in animals exposed to CS was significantly attenuated after pretreatment with SCFP-1. The cough suppression of SCFP-1 (500 mg/kg) stablly lasted during the whole 5 h of time-dependent experiment, while no positive effect was observed after 300 min of oral administration of codeine. CONCLUSIONS: SCFP-1 is one of the antitussive components of S. chinensis.

Effects of Schisandra chinensis extracts on cough and pulmonary inflammation in a cough hypersensitivity guinea pig model induced by cigarette smoke exposure.

Schisandra chinensis (S. chinensis) is a traditional Chinese medicine commonly used in prescription medications for the treatment of chronic cough. However, the material basis of S. chinensis in relieving cough has not been completely elucidated yet. This study established a guinea pig model of cough hypersensitivity induced by 14 days of cigarette smoke (CS) exposure, to evaluate the antitussive, antioxidant, and anti-inflammatory effects of three S. chinensis extracts. And then the function of four lignans in reducing expression of TRPV1 and TRPA1 was examined using A549 cells induced by cigarette smoke extract (CSE). The results demonstrated that both ethanol extract (EE) and ethanol-water extract (EWE) of S. chinensis, but not water extract (WE), significantly reduced the cough frequency enhanced by 0.4M citric acid solution in these cough hypersensitivity guinea pigs. Meanwhile, pretreatment with EE and EWE both significantly attenuated the CS-induced increase in infiltration of pulmonary neutrophils and total inflammatory cells, as well as pulmonary MDA, TNF-α, and IL-8, while remarkably increased activities of pulmonary SOD and GSH. According to H&E and immunofluorescence staining assays, airway epithelium hyperplasia, smooth muscle thickening, inflammatory cells infiltration, as well as expression of TRPV1 and TRPA1, were significantly attenuated in animals pretreatment with 1g/kg EE. Moreover, four lignans of EE, including schizandrin, schisantherin A, deoxyschizandrin and γ-schisandrin, significantly inhibited CSE-induced expression of TRPV1, TRPA1 and NOS3, as well as NO release in A549 cells. In conclusion, S. chinensis reduces cough frequency and pulmonary inflammation in the CS-induced cough hypersensitivity guinea pigs. Lignans may be the active components.

Reference values of induced sputum cytology in healthy children in guangzhou, southern china.

OBJECTIVE: To establish normal reference values of induced sputum cytology in healthy children in southern China. METHODS: During a period from January 2010 to December 2011, a total of 580 healthy children (5-16 years of age) were approached. A total of 266 children (137 boys and 129 girls) participated in the study. Sputum induction was carried out by using 5% hypertonic saline. Cell types in the sputum were examined by using routine methods. RESULTS: Sputum induction was completed in 175 of the 266 subjects (65.79%), but 16 sputum samples were disqualified. The overall success rate was 59.77% (159/266). Macrophages and neutrophils were the predominant cell types: macrophages: median, 76.14%; interquartile range (IQR), 32.68%; and 2.5% to 97.5% percentile, 1.00% to 94.50%; neutrophils: median, 20.67%; IQR, 33.0%; and 2.5% to 97.5% percentile, 4.00% to 92.75%; eosinophils: median, 0.39%; IQR, 1.93%; and 2.5% to 97.5% percentile, 0.00% to 6.50%; and lymphocytes: median, 1.22%; IQR, 2.04%; and 2.5% to 97.5% percentile, 0.00% to 5.00%. The cell types did not differ among different age, gender, and passive smoking groups. Adverse events occurred in 4.4% (7/159) of the participants who completed the procedures but required no specific treatment to dissipate. Peak expiratory flow did not differ between those who completed the procedures compared with those who did not, suggesting that the procedure is safe and feasible in children. CONCLUSIONS: The current study represents the first attempt to develop normal reference values of induced sputum cytology in Chinese children, and could be used as a control for future studies.

[Preliminary establishment of cytological examination and the normal reference values for hypertonic saline solution-induced sputum of healthy children in Guangzhou].

OBJECTIVE: To establish the method of cytological examination and the normal reference values for hypertonic saline solution-induced sputum of healthy children (age range from 5 to 15 years) with physical examination in Guangzhou. METHOD: A total of 352 children, 5 to 15 years old, were enrolled from primary school and middle school in Guangzhou from January to December, 2010. All subjects completed a standardized questionnaire on the presence of respiratory, allergic symptoms and family history, the medical history and the physical examination was performed by doctors, lung function (forced expiratory volume at 1 s in predicted normal, FEV(1)%) was determined. There were 266 healthy children (137 males, 129 females) who were selected and undergone hypertonic saline solution induction of sputum, and cytological examination was performed. Hypertonic saline (5%) was nebulized and inhaled for 15 - 30 min. No expectoration within 30 min was defined as failure, and the procedure was terminated. The part of opaque and higher density sputum samples was detected by cytology. The proportion of neutrophils, lymphocytes, eosinophils, macrophages and monocytes was calculated. This study was approved by the institutional Ethics Review Committee of First Affiliated Hospital of Guangzhou Medical College. Informed consent was obtained from the legal guardians of all participants following a detailed description of the purpose and potential benefits of the study. RESULT: There were 175 subjects' induced sputum specimens (175/266, 65.8%), non-qualified sputum samples were obtained from 16 of the subjects. The proportions of median (IQR) of lymphocytes were 0.012 (0.020), 95%CI were ranged from 0.015 to 0.022; neutrophils 0.207 (0.330), 95%CI 0.266 - 0.356 macrophages 0.761 (0.327), 95%CI 0.607 - 0.699; eosinophils 0.004 (0.019), 95%CI 0.013 - 0.022. There were no significant differences in proportions of cytological findings of female or male, different age groups and second-hand smoking or not (all P > 0.05). The incidence of adverse event was 4.40% (7/159). CONCLUSION: The method and the preliminary data may be used for research, diagnosis and treatment of patients with chronic cough and airway inflammation.

[Effects of repeated esophageal acid infusion on airway resistance and airway reactivity in guinea pigs and the mechanism].

OBJECTIVE: To observe the effect of repeated esophageal acid infusion on specific airway resistance (sRaw) and airway reactivity in the guinea pigs and explore the mechanism. METHODS: sRaw and airway reactivity were measured by double-chamber plethysmography in normal control group (group N), saline control group (group NS), and repeated acid irrigation group (group H). The initial measurement was used as the baseline sRaw and airway reactivity (1d1), and 2 h after the initial measurement, sRaw and airway reactivity were measured again (1d2). Similarly, such measurements were repeated on the 15th day for all the guinea pigs (15d1, 15d2) with a 2-h interval. The content of Substance P (SP) and vasoactive intestinal peptide (VIP) in lung tissue, trachea, BALF and ganglion were detected by ELISA. RESULTS: The percent change of sRaw, (15d2-1d1)/1d1 in group H was significantly higher than that in group N. The differences in the airway reactivity of the group N, group NS, and group H were not statistically significant. The SP content in the lung, trachea, ganglion and bronchoalveolar lavage fluid (BALF) in group H was significantly higher than those in group N. The SP content in ganglion showed a significant positive correlation to that in the trachea. No significant differences were found in the VIP content in the lung, trachea, ganglion or BALF between the groups. CONCLUSION: Repeated esophageal acid infusion increases the airway resistance, but not the airway reactivity in normal guinea pigs. SP may be involved in development of high sRaw through the esophageal-tracheobronchial reflex.

[The role of airway neurogenic inflammation in gastro-esophageal reflux induced cough].

OBJECTIVE: To investigate the role of airway neurogenic inflammation in the pathogenesis of gastro-esophageal reflux induced cough (GERC). METHODS: Sputum was induced by hypertonic saline aerosol inhalation in 20 patients with GERC (GERC group), 10 healthy subjects (normal control group) and 8 patients with chronic cough due to other causes but complicated with gastro-esophageal reflux diseases (GERD, GERD group). Airway mucosal biopsy was performed in 6 patients with GERC and 4 patients with GERD using flexible fiberoptic bronchoscopy. The expression of substance P (SP), neurokinin 1 receptor and neurokinin A (NKA) in sputum cells and airway mucosa were detected by immunohistochemistry, and was assessed semi-quantitatively. SP, NKA, and NKB in the supernatant of induced sputum were measured with enzyme linked immunosorbent assay. Calcitonin gene-related peptide (CGRP) was measured with radioimmunoassay. RESULTS: The concentration of SP in the supernatant of induced sputum was significantly higher in GERC group [(266 +/- 207) ng/L] than those in normal control group [(143 +/- 36) ng/L, P < 0.05] and GERD group [(130 +/- 11) ng/L, P < 0.05], and the sputum supernatant concentration of CGRP in GERC group [(180 +/- 83) ng/L] was significantly higher than those in normal control group [(105 +/- 64) ng/L, P < 0.01] and GERD group [(89 +/- 16) ng/L, P < 0.01]. The expression of SP, NK-1 receptor and NKA in induced sputum cells in GERC group were significantly higher than those in normal control group (P < 0.01, < 0.05, < 0.05) and GERD group (all P < 0.05); Expressions of SP in airway mucosa was significantly higher in GERC group than in GERD group (P < 0.01). After treatment, the concentration of CGRP in the supernatant of sputum in GERC patients was significantly lower than that before treatment (P < 0.05); the expression of SP, NK-1 and NKA in the induced sputum cells were significantly lower than that before treatment (P < 0.01, P < 0.01 or P < 0.05). CONCLUSION: There is airway neurogenic inflammation in GERC patients, which maybe closely related to the development of GERC.