Enhanced inflammasome activation and reduced sphingosine-1 phosphate S1P signalling in a respiratory mucoobstructive disease model

Background:Inflammasomes and sphingosine-1-phosphate (S1P) signalling are increasingly subject to intensive research in human diseases. We hypothesize that in respiratory muco-obstructive diseases, mucus obstruction enhances NLRP3 inflammasome activation and dysregulated S1P signalling. Methods:Lung tissues from mice overexpressing the beta-unit of the epithelial sodium channel (βENaC) and their littermate controls were examined by histology, immunofluorescence and confocal microscopy, followed by ImageJ quantitative analysis. Results:Lower airways in βENaC mice showed patchy patterns of mucus obstruction and neutrophil-dominant infiltrations. In contrast to a ubiquitous distribution of TNFα specks, significantly (p < 0.05) increased specks of bronchiolar NLRP3, IL-1β, and IgG in the βENaC mouse lungs were localized to the vicinity of mucus obstruction sites. Bright Spinster homologue 2 (SPNS2) at the epithelial apex and positive correlation with sphingosine kinase 1 (SPHK1) (R2 = 0.640; p < 0.001) supported the normal bronchial epithelium as an active generator of extracellular S1P. SPNS2 in βENaC mice was sharply reduced (38%, p < 0.05) and lost apical localization at sites of mucus obstruction. A significant (34%; p < 0.01) decrease in epithelial SPHK2 was also noted at mucus obstruction sites. Conclusion:These results support that mucus obstruction may enhance NLRP3 inflammasome activation and dysregulated S1P signaling

Organotypic sinonasal airway culture systems are predictive of the mucociliary phenotype produced by bronchial airway epithelial cells

Published online: 10 November 2022Differentiated air-liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models. Basal progenitor AEC were isolated from healthy participants using a cytological brushing method and differentiated into epithelia on transwells until the mucociliary phenotype was observed. Tissue architecture was assessed using H&E and alcian blue/Verhoeff-Van Gieson staining, immunofluorescence (for cilia via acetylated α-tubulin labelling) and scanning electron microscopy. Differentiation and the formation of tight-junctions were monitored over the culture period (day 1-32) by quantifying trans-epithelial electrical resistance. End point (day 32) tight junction protein expression was assessed using Western blot analysis of ZO-1, Occludin-1 and Claudin-1. Reverse transcription qPCR-array was used to assess immunomodulatory and autophagy-specific transcript profiles. All outcome measures were assessed using R-statistical software. Mucociliary architecture was comparable for nAEC and bAEC-derived cultures, e.g. cell density P = 0.55, epithelial height P = 0.88 and cilia abundance P = 0.41. Trans-epithelial electrical resistance measures were distinct from day 1-14, converged over days 16-32, and were statistically similar over the entire culture period (global P  0.05). Transcript analysis for inflammatory markers demonstrated significant variation between nAEC and bAEC epithelial cultures, and favoured increased abundance in the nAEC model (e.g. TGFβ and IL-1β; P < 0.05). Conversely, the abundance of autophagy-related transcripts were comparable and the range of outcome measures for either model exhibited a considerably more confined uncertainty distribution than those observed for the inflammatory markers. Organotypic air-liquid interface models of nAEC are predictive of outcomes related to barrier function, mucociliary architecture and autophagy gene activity in corresponding bAEC models. However, inflammatory markers exhibited wide variation which may be explained by the sentinel immunological surveillance role of the sinonasal epithelium.Juliette Delhove, Moayed Alawami, Matthew Macowan, Susan E. Lester, Phan T. Nguyen, Hubertus P. A. Jersmann, Paul N. Reynolds and Eugene Rosciol

Interventional low-dose azithromycin attenuates cigarette smoke-induced emphysema and lung inflammation in mice

Cigarette smoke (CS)-induced emphysema is an important contributor to chronic obstructive pulmonary disease (COPD). We have shown the efficacy of azithromycin in reducing airway inflammation in COPD and in reducing exacerbations in severe asthma; however, the effects of long-term azithromycin on emphysema development have not been shown. We employed live animal imaging to monitor emphysema-like development and the effects of interventional azithromycin treatment in CS-exposed mice. BALB/c mice (female, 10 weeks; n = 10) were exposed to CS for 1 hr twice daily, 5 days/week, and for 12 weeks (CS). Half were cotreated with low-dose azithromycin during weeks 7-12 (CS + Azi; 0.2 mg kg-1  day-1 ). Microcomputed tomography (CT) and magnetic resonance imaging (MRI) scans were acquired longitudinally. Histological examinations were performed post mortem (mean linear intercept (Lm) and leukocyte infiltration). CS increased median Lm (CS: 42.45 µm versus control: 34.7 µm; p = .0317), this was recovered in CS + Azi mice (33.03 µm). Average CT values were reduced in CS mice (CS: -399.5 Hounsfield units (HU) versus control: -384.9 HU; p = .0286) but not in CS + Azi mice (-377.3 HU). CT values negatively correlated with Lm (r = -.7972; p = .0029) and T2 -weighted MRI (r = -.6434; p = .0278). MRI also showed significant CS-induced inflammatory changes that were attenuated by azithromycin in the lungs, and positively correlated with Lm (r = .7622; p = .0055) and inflammatory foci counts (r = .6503; p = .0257). Monitoring of emphysema development is possible via micro-CT and MRI. Interventional azithromycin treatment in CS-exposed mice attenuated the development of pulmonary emphysema-like changes.Matthew G. Macowan, Hong Liu, Miranda Ween, Rhys Hamon, Hai B. Tran Sandra Hodge ... et al

Early life inter-kingdom interactions shape the immunological environment of the airways

Background: There is increasing evidence that the airway microbiome plays a key role in the establishment of respiratory health by interacting with the developing immune system early in life. While it has become clear that bacteria are involved in this process, there is a knowledge gap concerning the role of fungi. Moreover, the inter-kingdom interactions that influence immune development remain unknown. In this prospective exploratory human study, we aimed to determine early post-natal microbial and immunological features of the upper airways in 121 healthy newborns. Results: We found that the oropharynx and nasal cavity represent distinct ecological niches for bacteria and fungi. Breastfeeding correlated with changes in microbiota composition of oropharyngeal samples with the greatest impact upon the relative abundance of Streptococcus species and Candida. Host transcriptome profiling revealed that genes with the highest expression variation were immunological in nature. Multi-omics factor analysis of host and microbial data revealed unique co-variation patterns. Conclusion: These data provide evidence of a diverse multi-kingdom microbiota linked with local immunological characteristics in the first week of life that could represent distinct trajectories for future respiratory health

The skin microbiome in the first year of life and its association with atopic dermatitis

publishedVersio

Vocational facilities in Indiana.

The purpose of the study was to measure and analyze accessibility of area vocational facilities in Indiana to physically disabled persons. Empirical data did not exist to determine accessibility of area vocational facilities in Indiana. The vocational facilities were all self-contained, or not attached to comprehensive high schools.Federal fiscal support of vocational education had been constant beginning with the Morrill Act of 1862. Intent of federal legislation toward accessibility and vocational education was to insure any person, regardless of handicap, an unobstructed, free and appropriate education.Enrollment of secondary vocational education students in Indiana has increased over ninety percent since 1967. Vocational schools' receiving federal monetary support have mandated accessibility for handicapped persons. Therefore, it is important that existing vocational facilities maintain accessibility compliance and future buildings be designed with accessibility for handicapped persons kept paramount.Twenty-two area vocational facilities were visited and surveyed between December, 1981 and March, 1982. The instrument used to survey vocational facilities was The Revised Accessibility Checklist as recommended by the Iowa Chapter of the American Institute of Architects.The Revised Accessibility Checklist was based on American National Standards Institute Specifications 117.1. ANSI Specification 117.1 was the established standard used in determining compliance with the Rehabilitation Act of 1973. The Rehabilitation Act of 1973 required all buildings receiving federal funds be accessible to physically handicapped persons. Section 504 of the Rehabilitation Act of 1973 required structures to be in compliance by June, 1980.Individual vocational facilities were evaluated according to specific accessibility checklist criteria. Classifications in determining individual building accessibility were Excellent, Good, Average, Below Average, and Poor. None was judged excellent, four were good, none was average, fourteen were below average and four were poor.Conclusions based on the findings and data were:The majority of facilities were not accessible to physically disabled persons. 2) Federal legislation mandating accessibility has had limited effect. 3) Facilities constructed after 1975 were in substantial compliance with accessibility standards. 4) Parking lots, restrooms, water fountains and telephones could be feasibly altered to comply with accessibility standards.Recommendations for specific renovations to the twenty-two facilities surveyed were made for improving accessibility.Thesis (D. Ed.

Cigarette smoke-induced emphysema and lung inflammation are attenuated by low-dose azithromycin in BALB/c mice

Abstract published in TSANZ20 Posters AbstractsMacowan, M, Liu, H, Keller, M, Ween, M, Hamon, R, Tran, H, Hodge,