Antibiotic-driven dysbiosis in early life disrupts indole-3-propionic acid production and exacerbates allergic airway inflammation in adulthood.

Antibiotic use in early life disrupts microbial colonization and increases the risk of developing allergies and asthma. We report that mice given antibiotics in early life (EL-Abx), but not in adulthood, were more susceptible to house dust mite (HDM)-induced allergic airway inflammation. This susceptibility was maintained even after normalization of the gut microbiome. EL-Abx decreased systemic levels of indole-3-propionic acid (IPA), which induced long-term changes to cellular stress, metabolism, and mitochondrial respiration in the lung epithelium. IPA reduced mitochondrial respiration and superoxide production and altered chemokine and cytokine production. Consequently, early-life IPA supplementation protected EL-Abx mice against exacerbated HDM-induced allergic airway inflammation in adulthood. These results reveal a mechanism through which EL-Abx can predispose the lung to allergic airway inflammation and highlight a possible preventative approach to mitigate the detrimental consequences of EL-Abx.

Deep multiomic profiling reveals molecular signatures that underpin preschool wheeze and asthma.

BACKGROUND: Wheezing in childhood is prevalent, with over one-half of all children experiencing at least 1 episode by age 6. The pathophysiology of wheeze, especially why some children develop asthma while others do not, remains unclear. OBJECTIVES: This study addresses the knowledge gap by investigating the transition from preschool wheeze to asthma using multiomic profiling. METHODS: Unsupervised, group-agnostic integrative multiomic factor analysis was performed using host/bacterial (meta)transcriptomic and bacterial shotgun metagenomic datasets from bronchial brush samples paired with metabolomic/lipidomic data from bronchoalveolar lavage samples acquired from children 1-17 years old. RESULTS: Two multiomic factors were identified: one characterizing preschool-aged recurrent wheeze and another capturing an inferred trajectory from health to wheeze and school-aged asthma. Recurrent wheeze was driven by type 1-immune signatures, coupled with upregulation of immune-related and neutrophil-associated lipids and metabolites. Comparatively, progression toward asthma from ages 1 to 18 was dominated by changes related to airway epithelial cell gene expression, type 2-immune responses, and constituents of the airway microbiome, such as increased Haemophilus influenzae. CONCLUSIONS: These factors highlighted distinctions between an inflammation-related phenotype in preschool wheeze, and the predominance of airway epithelial-related changes linked with the inferred trajectory toward asthma. These findings provide insights into the differential mechanisms driving the progression from wheeze to asthma and may inform targeted therapeutic strategies.

The longitudinal microbial and metabolic landscape of infant cystic fibrosis: the gut-lung axis.

BACKGROUND AND AIM: In cystic fibrosis, gastrointestinal dysfunction and lower airway infection occur early and are independently associated with poorer outcomes in childhood. This study aimed to define the relationship between the microbiota at each niche during the first 2 years of life, its association with growth and airway inflammation, and explanatory features in the metabolome. MATERIALS AND METHODS: 67 bronchoalveolar lavage fluid (BALF), 62 plasma and 105 stool samples were collected from 39 infants with cystic fibrosis between 0 and 24 months who were treated with prophylactic antibiotics. 16S rRNA amplicon and shotgun metagenomic sequencing were performed on BALF and stool samples, respectively; metabolomic analyses were performed on all sample types. Sequencing data from healthy age-matched infants were used as controls. RESULTS: Bacterial diversity increased over the first 2 years in both BALF and stool, and microbial maturation was delayed in comparison to healthy controls from the RESONANCE cohort. Correlations between their respective abundance in both sites suggest stool may serve as a noninvasive alternative for detecting BALF Pseudomonas and Veillonella. Multisite metabolomic analyses revealed age- and growth-related changes, associations with neutrophilic airway inflammation, and a set of core systemic metabolites. BALF Pseudomonas abundance was correlated with altered stool microbiome composition and systemic metabolite alterations, highlighting a complex gut-plasma-lung interplay and new targets with therapeutic potential. CONCLUSION: Exploration of the gut-lung microbiome and metabolome reveals diverse multisite interactions in cystic fibrosis that emerge in early life. Gut-lung metabolomic links with airway inflammation and Pseudomonas abundance warrant further investigation for clinical utility, particularly in non-expectorating patients.

Modelling lung infection with Klebsiella pneumoniae after murine traumatic brain injury.

Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated with increased morbidity and mortality. In this study, we established a model of intratracheal Klebsiella pneumoniae administration in young adult male and female mice, at 4 days following an experimental TBI, to investigate how K. pneumoniae infection influences acute post-TBI outcomes. A dose-response curve determined the optimal dose of K. pneumoniae for inoculation (1 x 10^6 colony forming units), and administration at 4 days post-TBI resulted in transient body weight loss and sickness behaviors (hypoactivity and acute dyspnea). K. pneumoniae infection led to an increase in pro-inflammatory cytokines in serum and bronchoalveolar lavage fluid at 24 h post-infection, in both TBI and sham (uninjured) mice. By 7 days, when myeloperoxidase + neutrophil numbers had returned to baseline in all groups, lung histopathology was observed with an increase in airspace size in TBI + K. pneumoniae mice compared to TBI + vehicle mice. In the brain, increased neuroinflammatory gene expression was observed acutely in response to TBI, with an exacerbated increase in Ccl2 and Hmox1 in TBI + K. pneumoniae mice compared to either TBI or K. pneumoniae alone. However, the presence of neuroinflammatory immune cells in the injured brain, and the extent of damage to cortical and hippocampal brain tissue, was comparable between K. pneumoniae and vehicle-treated mice by 7 days. Examination of the fecal microbiome across a time course did not reveal any pronounced effects of either injury or K. pneumoniae on bacterial diversity or abundance. Together, these findings demonstrate that K. pneumoniae lung infection after TBI induces an acute and transient inflammatory response, primarily localized to the lungs with some systemic effects. However, this infection had minimal impact on secondary injury processes in the brain following TBI. Future studies are needed to evaluate the potential longer-term consequences of this dual-hit insult.

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

BACKGROUND: Early-life microbial colonization of the skin may modulate the immune system and impact the development of atopic dermatitis (AD) and allergic diseases later in life. To address this question, we assessed the association between the skin microbiome and AD, skin barrier integrity and allergic diseases in the first year of life. We further explored the evolution of the skin microbiome with age and its possible determinants, including delivery mode. METHODS: Skin microbiome was sampled from the lateral upper arm on the first day of life, and at 3, 6, and 12 months of age. Bacterial communities were assessed by 16S rRNA gene amplicon sequencing in 346 infants from the PreventADALL population-based birth cohort study, representing 970 samples. Clinical investigations included skin examination and skin barrier function measured as trans-epidermal water loss (TEWL) at the site and time of microbiome sampling at 3, 6, and 12 months. Parental background information was recorded in electronic questionnaires, and delivery mode (including vaginal delivery (VD), VD in water, elective caesarean section (CS) and emergency CS) was obtained from maternal hospital charts. RESULTS: Strong temporal variations in skin bacterial community composition were found in the first year of life, with distinct patterns associated with different ages. Confirming our hypothesis, skin bacterial community composition in the first year of life was associated with skin barrier integrity and later onsets of AD. Delivery mode had a strong impact on the microbiome composition at birth, with each mode leading to distinct patterns of colonization. Other possible determinants of the skin microbiome were identified, including environmental and parental factors as well as breastfeeding. CONCLUSION: Skin microbiome composition during infancy is defined by age, transiently influenced by delivery mode as well as environmental, parental factors and breastfeeding. The microbiome is also associated with skin barrier integrity and the onset of AD.

Effects of E-cigarette E-liquid components on bronchial epithelial cells: Demonstration of dysfunctional efferocytosis.

BACKGROUND AND OBJECTIVE: E-cigarettes are often marketed and thought of as emitting harmless vapour; however, verification of their safety for non-smokers is scarce. We have previously shown that E-cigarettes cause decreased phagocytosis of bacteria by macrophages via reductions in surface bacterial recognition receptors. This study assessed the effect of E-cigarette constituents, 3 E-liquid apple flavours, nicotine, vegetable glycerine and propylene glycol, on bronchial epithelial cell viability, apoptosis and cytokine secretion and macrophage phagocytosis of apoptotic airway cells and phagocytic recognition molecules. METHODS: Cell necrosis and apoptosis were measured by Sytox Green stain and Annexin V. Efferocytosis was measured by internalization of pHrodo Green labelled apoptotic airway cells by macrophages. Expression of macrophage cell surface apoptotic cell receptors was measured by flow cytometry. Cytokine release by E-cigarette-exposed airway cells was measured by cytokine bead array. RESULTS: E-cigarette vapour increased primary bronchial epithelial necrosis and apoptosis. E-cigarette vapour reduced efferocytosis (lowest flavour 12.1%) versus control (20.2%, P = 0.032). The efferocytosis receptor CD44 was reduced by one flavour (MFI 1863 vs 2332 control, P = 0.016) and all components reduced expression of CD36, including the glycol bases (MFI 1067-12 274 vs 1415 control). Reduced secretion of TNF-α, IL-6, IP-10, MIP-1α and MIP-1ß was observed for all flavour variants. CONCLUSION: E-cigarettes can cause bronchial epithelial apoptosis and macrophage efferocytosis dysfunction via reduced expression of apoptotic cell recognition receptors. These data further show that E-cigarettes should not be considered harmless to non-smokers and their effects may go far beyond cytotoxicity to cells.

Multi-omics integration reveals a nonlinear signature that precedes progression of lung fibrosis.

Objectives: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive interstitial lung disease with poor outcomes. While decades of research have shed light on pathophysiological mechanisms associated with the disease, our understanding of the early molecular events driving IPF and its progression is limited. With this study, we aimed to model the leading edge of fibrosis using a data-driven approach. Methods: Multiple omics modalities (transcriptomics, metabolomics and lipidomics) of healthy and IPF lung explants representing different stages of fibrosis were combined using an unbiased approach. Multi-Omics Factor Analysis of datasets revealed latent factors specifically linked with established fibrotic disease (Factor1) and disease progression (Factor2). Results: Features characterising Factor1 comprised well-established hallmarks of fibrotic disease such as defects in surfactant, epithelial-mesenchymal transition, extracellular matrix deposition, mitochondrial dysfunction and purine metabolism. Comparatively, Factor2 identified a signature revealing a nonlinear trajectory towards disease progression. Molecular features characterising Factor2 included genes related to transcriptional regulation of cell differentiation, ciliogenesis and a subset of lipids from the endocannabinoid class. Machine learning models, trained upon the top transcriptomics features of each factor, accurately predicted disease status and progression when tested on two independent datasets. Conclusion: This multi-omics integrative approach has revealed a unique signature which may represent the inflection point in disease progression, representing a promising avenue for the identification of therapeutic targets aimed at addressing the progressive nature of the disease.

Gut instinct: Sex differences in the gut microbiome are associated with changes in adolescent nociception following maternal separation in rats.

Adolescent chronic pain is a growing public health epidemic. Our understanding of its etiology is limited; however, several factors can increase susceptibility, often developing in response to an acute pain trigger such as a surgical procedure or mild traumatic brain injury (mTBI), or an adverse childhood experience (ACE). Additionally, the prevalence and manifestation of chronic pain is sexually dimorphic, with double the rates in females than males. Despite this, the majority of pre-clinical pain research focuses on males, leaving a gap in mechanistic understanding for females. Given that emerging evidence has linked the gut microbiome and the brain-gut-immune axis to various pain disorders, we aimed to investigate sex-dependent changes in taxonomic and functional gut microbiome features following an ACE and acute injury as chronic pain triggers. Male and female Sprague Dawley rat pups were randomly assigned to either a maternal separation (MS) or no stress paradigm, then further into a sham, mTBI, or surgery condition. Chronically, the von Frey test was used to measure mechanical nociception, and fecal samples were collected for 16S rRNA sequencing. Animals in the surgery group had an increase in pain sensitivity when compared to mTBI and sham groups, and this was complemented by changes to the gut microbiome. In addition, significant sex differences were identified in gut microbiome composition, which were exacerbated in response to MS. Overall, we provide preliminary evidence for sex differences and ACE-induced changes in bacterial composition that, when combined, may be contributing to heterogeneity in pain outcomes.

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

Differentiated 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 < 0.001). This agreed with end-point (day 32) measures of tight junction protein abundance which were non-significant for each analyte (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. TGFß and IL-1ß; 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.

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. TRIAL REGISTRATION: NHS Health Research Authority, IRAS ID 199053. Registered 5 Oct 2016. https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/breathing-together/ Video abstract.

The role of oxidised self-lipids and alveolar macrophage CD1b expression in COPD.

In chronic obstructive pulmonary disease (COPD) apoptotic bronchial epithelial cells are increased, and their phagocytosis by alveolar macrophages (AM) is decreased alongside bacterial phagocytosis. Epithelial cellular lipids, including those exposed on uncleared apoptotic bodies, can become oxidized, and may be recognized and presented as non-self by antigen presenting cells. CD1b is a lipid-presenting protein, previously only described in dendritic cells. We investigated whether CD1b is upregulated in COPD AM, and whether lipid oxidation products are found in the airways of cigarette smoke (CS) exposed mice. We also characterise CD1b for the first time in a range of macrophages and assess CD1b expression and phagocytic function in response to oxidised lipid. Bronchoalveolar lavage and exhaled breath condensate were collected from never-smoker, current-smoker, and COPD patients and AM CD1b expression and airway 8-isoprostane levels assessed. Malondialdehyde was measured in CS-exposed mouse airways by confocal/immunofluorescence. Oxidation of lipids produced from CS-exposed 16HBE14o- (HBE) bronchial epithelial cells was assessed by spectrophotometry and changes in lipid classes assessed by mass spectrometry. 16HBE cell toxicity was measured by flow cytometry as was phagocytosis, CD1b expression, HLA class I/II, and mannose receptor (MR) in monocyte derived macrophages (MDM). AM CD1b was significantly increased in COPD smokers (4.5 fold), COPD ex-smokers (4.3 fold), and smokers (3.9 fold), and AM CD1b significantly correlated with disease severity (FEV1) and smoking pack years. Airway 8-isoprostane also increased in smokers and COPD smokers and ex-smokers. Malondialdehyde was significantly increased in the bronchial epithelium of CS-exposed mice (MFI of 18.18 vs 23.50 for control). Oxidised lipid was produced from CS-exposed bronchial epithelial cells (9.8-fold of control) and showed a different overall lipid makeup to that of control total cellular lipid. This oxidised epithelial lipid significantly upregulated MDM CD1b, caused bronchial epithelial cell toxicity, and reduced MDM phagocytic capacity and MR in a dose dependent manner. Increased levels of oxidised lipids in the airways of COPD patients may be responsible for reduced phagocytosis and may become a self-antigen to be presented by CD1b on macrophages to perpetuate disease progression despite smoking cessation.

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 (ßENaC) and their littermate controls were examined by histology, immunofluorescence and confocal microscopy, followed by ImageJ quantitative analysis. RESULTS: Lower airways in ßENaC 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-1ß, and IgG in the ßENaC 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 ßENaC 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.

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.

Sphingosine signaling dysfunction in airway cells as a potential contributor to progression from protracted bacterial bronchitis to bronchiectasis in children.

AIM: Protracted bacterial bronchitis (PBB) is considered a potential precursor to bronchiectasis (BE) in some children. We previously showed that alveolar macrophages (AM) from children with PBB or BE have a similar significant defect in phagocytic capacity, with proinflammatory associations. We hypothesized that the mechanisms responsible for this defect involve dysregulation of the sphingosine-1-phosphate (S1P) signaling pathway, as we have found in adult inflammatory lung diseases. METHOD: We employed a Custom TaqMan OpenArray to investigate gene expression of S1P-generating enzymes: sphingosine kinases (SPHK) 1/2, S1P phosphatase 2 (SGPP2), S1P lyase 1 (SGPL1), S1P receptors (S1PR) 1/2/4/5; proinflammatory cytokines TNF-α (TNF) and IFNγ (IFNG), the cytotoxic mediator granzyme B (GZMB), and inflammasomes AIM2 and NLRP3, in bronchoalveolar lavage from 15 children with BE, 15 with PBB and 17 age-matched controls, and determined association with clinical/demographic variables and airway inflammation. RESULT: Significantly increased expression of S1PR1, S1PR2, and SPHK1 was noted in PBB and BE AM vs controls with increased SGPP2 only in PBB. TNF, IFNG, AIM2, and NLRP3 were significantly increased in both disease groups with increased GZMB only in PBB. There were no significant differences in the expression of any other S1P-related mediator between groups. There were significant positive associations between Haemophilus influenzae growth and expression of S1PR1 and NLRP3; between S1PR1 and S1PR2, NLRP3 and IFNG; between S1PR2 and AIM2, SPHK1, and SPHK2; and between SPHK1 and GZMB, IFNG, AIM2, and NLRP3. CONCLUSION: Children with PBB and BE share similar S1P-associated gene expression profiles. AM phagocytic dysfunction and inflammation in these children may occur due to dysregulated S1P signaling.