Inflammatory profiles in sputum and blood of people with TB with and without HIV coinfection.

Although tuberculosis (TB) remains a major killer among infectious diseases and the leading cause of death for people with HIV, drivers of immunopathology, particularly at the site of infection in the lungs remain incompletely understood. To fill this gap, we compared cytokine profiles in paired plasma and sputum samples collected from adults with pulmonary TB with and without HIV. We found that people with pulmonary TB with HIV had significantly higher markers of inflammation in both plasma and sputum than those without HIV; these differences were present despite a similar extent of radiographic involvement. We also found that the strength and direction of correlations between biomarkers in the blood and lung compartments differed by HIV status and people with HIV had more positive correlations than those without HIV. Future studies can further explore these differences in inflammation by HIV status across the blood and lung compartments and seek to establish how these profiles may be associated with long-term outcomes and lung health after completion of TB treatment.

Why Certain Repurposed Drugs Are Unlikely to Be Effective Antivirals to Treat SARS-CoV-2 Infections.

Most repurposed drugs have proved ineffective for treating COVID-19. We evaluated median effective and toxic concentrations (EC50, CC50) of 49 drugs, mostly from previous clinical trials, in Vero cells. Ratios of reported unbound peak plasma concentrations, (Cmax)/EC50, were used to predict the potential in vivo efficacy. The 20 drugs with the highest ratios were retested in human Calu-3 and Caco-2 cells, and their CC50 was determined in an expanded panel of cell lines. Many of the 20 drugs with the highest ratios were inactive in human Calu-3 and Caco-2 cells. Antivirals effective in controlled clinical trials had unbound Cmax/EC50 ≥ 6.8 in Calu-3 or Caco-2 cells. EC50 of nucleoside analogs were cell dependent. This approach and earlier availability of more relevant cultures could have reduced the number of unwarranted clinical trials.

OMIP-100: A flow cytometry panel to investigate human neutrophil subsets.

This 14-color, 13-antibody optimized multicolor immunofluorescence panel (OMIP) was designed for deep profiling of neutrophil subsets in various types of human samples to contextualize neutrophil plasticity in a range of healthy and diseased states. Markers present in the OMIP allow the profiling of neutrophil subsets associated with ontogeny, migration, phagocytosis capacity, granule release, and immune modulation. For panel design, we ensured that the commonly available fluorophores FITC/AF488, PE, and APC were assigned to the intracellular subset marker Olfactomedin 4, the maturity and activation marker CD10, and whole blood subset marker CD177, respectively. These markers can be easily replaced without affecting the core identification of neutrophils, enabling antibodies to new neutrophil antigens of interest or for fluorescent substrates to assess different neutrophil functions to be easily explored. Panel optimization was performed on whole blood and purified neutrophils. We demonstrate applications on clinical samples (whole blood and saliva) and experimental endpoints (purified neutrophils stimulated through an in vitro transmigration assay). We hope that providing a uniform platform to analyze neutrophil plasticity in various sample types will facilitate the future understanding of neutrophil subsets in health and disease.

Key inflammatory markers in bronchoalveolar lavage predict bronchiectasis progression in young children with CF.

INTRODUCTION: Inflammation appears early in cystic fibrosis (CF) pathogenesis, with specific elevated inflammatory markers in bronchoalveolar lavage fluid (BALF) correlating with structural lung disease. Our aim was to identify markers of airway inflammation able to predict bronchiectasis progression over two years with high sensitivity and specificity. METHODS: Children with CF with two chest computed tomography (CT) scans and bronchoscopies at a two-year interval were included (n= 10 at 1 and 3 years and n= 27 at 3 and 5 years). Chest CTs were scored for increase in bronchiectasis (Δ%Bx), using the PRAGMA-CF score. BALF collected with the first CT scan were analyzed for neutrophil% (n= 36), myeloperoxidase (MPO) (n= 25), neutrophil elastase (NE) (n= 26), and with a protein array for inflammatory and fibrotic markers (n= 26). RESULTS: MPO, neutrophil%, and inducible T-cell costimulator ligand (ICOSLG), but not clinical characteristics, correlated significantly with Δ%Bx. Evaluation of neutrophil%, NE, MPO, interleukin-8 (IL-8), ICOSLG, and hepatocyte growth factor (HGF), for predicting an increase of > 0.5% of Δ%Bx in two years, showed that IL-8 had the best sensitivity (82%) and specificity (73%). Neutrophil%, ICOSLG and HGF had sensitivities of 85, 82, and 82% and specificities of 59, 67 and 60%, respectively. The odds ratio for risk of >0.5% Δ%Bx was higher for IL-8 (12.4) than for neutrophil%, ICOSLG, and HGF (5.9, 5.3, and 6.7, respectively). Sensitivity and specificity were lower for NE and MPO). CONCLUSIONS: High levels of IL-8, neutrophil%, ICOSGL and HGF in BALF may be good predictors for progression of bronchiectasis in young children with CF.

Stably-Inverted Apical-Out Human Upper Airway Organoids for SARS-CoV-2 Infection and Therapeutic Testing.

Apical-out organoids produced through eversion triggered by extra-organoid extracellular matrix (ECM) removal or degradation are generally small, structurally variable, and limited for viral infection and therapeutics testing. This work describes ECM-encapsulating, stably-inverted apical-out human upper airway organoids (AORBs) that are large (~500 µm diameter), consistently spherical, recapitulate in vivo-like cellular heterogeneity, and maintain their inverted morphology for over 60 days. Treatment of AORBs with IL-13 skews differentiation towards goblet cells and the apical-out geometry allows extra-organoid mucus collection. AORB maturation for 14 days induces strong co-expression of ACE2 and TMPRSS2 to allow high-yield infection with five SARS-CoV-2 variants. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds [remdesivir, bemnifosbuvir (AT-511), and nirmatrelvir] shows AORB antiviral assays to be comparable to gold-standard air-liquid interface cultures, but with higher throughput (~10-fold) and fewer cells (~100-fold). While this work focuses on SARS-CoV-2 applications, the consistent AORB shape and size, and one-organoid-per-well modularity broadly impacts in vitro human cell model standardization efforts in line with economic imperatives and recently updated FDA regulation on therapeutic testing.

Nasal airway inflammatory responses and pathogen detection in infants with cystic fibrosis.

BACKGROUND: Detecting airway inflammation non-invasively in infants with cystic fibrosis (CF) is difficult. We hypothesized that markers of inflammation in CF [IL-1ß, IL-6, IL-8, IL-10, IL-17A, neutrophil elastase (NE) and tumor necrosis factor (TNF-α)] could be measured in infants with CF from nasal fluid and would be elevated during viral infections or clinician-defined pulmonary exacerbations (PEx). METHODS: We collected nasal fluid, nasal swabs, and hair samples from 34 infants with CF during monthly clinic visits, sick visits, and hospitalizations. Nasal fluid was isolated and analyzed for cytokines. Respiratory viral detection on nasal swabs was performed using the Luminex NxTAG® Respiratory Pathogen Panel. Hair samples were analyzed for nicotine concentration by reverse-phase high-performance liquid chromatography. We compared nasal cytokine concentrations between the presence and absence of detected respiratory viruses, PEx, and smoke exposure. RESULTS: A total of 246 samples were analyzed. Compared to measurements in the absence of respiratory viruses, mean concentrations of IL-6, IL-8, TNF-α, and NE were significantly increased while IL-17A was significantly decreased in infants positive for respiratory viruses. IL-17A was significantly decreased and NE increased in those with a PEx. IL-8 and NE were significantly increased in infants with enteric pathogen positivity on airway cultures, but not P. aeruginosa or S. aureus. Compared to those with no smoke exposure, there were significantly higher levels of IL-6, IL-10, and NE in infants with detectable levels of nicotine. CONCLUSIONS: Noninvasive collection of nasal fluid may identify inflammation in infants with CF during changing clinical or environmental exposures.

Pulmonary exacerbations in early cystic fibrosis lung disease are marked by strong modulation of CD3 and PD-1 on luminal T cells.

Background: In chronic cystic fibrosis (CF) lung disease, neutrophilic inflammation and T-cell inhibition occur concomitantly, partly due to neutrophil-mediated release of the T-cell inhibitory enzyme Arg1. However, the onset of this tonic inhibition of T cells, and the impact of pulmonary exacerbations (PEs) on this process, remain unknown. Methods: Children with CF aged 0-5 years were enrolled in a longitudinal, single-center cohort study. Blood (n = 35) and bronchoalveolar lavage (BAL) fluid (n = 18) were collected at stable outpatient clinic visits or inpatient PE hospitalizations and analyzed by flow cytometry (for immune cell presence and phenotype) and 20-plex chemiluminescence assay (for immune mediators). Patients were categorized by PE history into (i) no prior PE, (ii) past history of PE prior to stable visit, or (iii) current PE. Results: PEs were associated with increased concentration of both pro- and anti-inflammatory mediators in BAL, and increased neutrophil frequency and G-CSF in circulation. PE BAL samples showed a trend toward an increased frequency of hyperexocytic "GRIM" neutrophils, which we previously identified in chronic CF. Interestingly, expression levels of the T-cell receptor associated molecule CD3 and of the inhibitory programmed death-1 (PD-1) receptor were respectively decreased and increased on T cells from BAL compared to blood in all patients. When categorized by PE status, CD3 and PD-1 expression on blood T cells did not differ among patients, while CD3 expression was decreased, and PD-1 expression was increased on BAL T cells from patients with current PE. Conclusions: Our findings suggest that airway T cells are engaged during early-life PEs, prior to the onset of chronic neutrophilic inflammation in CF. In addition, increased blood neutrophil frequency and a trend toward increased BAL frequency of hyperexocytic neutrophils suggest that childhood PEs may progressively shift the balance of CF airway immunity towards neutrophil dominance.

Airway macrophages display decreased expression of receptors mediating and regulating scavenging in early cystic fibrosis lung disease.

Background: Cystic fibrosis (CF) airway disease is characterized by chronic inflammation, featuring neutrophil influx to the lumen. Airway macrophages (AMs) can promote both inflammation and resolution, and are thus critical to maintaining and restoring homeostasis. CF AM functions, specifically scavenging activity and resolution of inflammation, have been shown to be impaired, yet underlying processes remain unknown. We hypothesized that impaired CF AM function results from an altered expression of receptors that mediate or regulate scavenging, and set out to investigate changes in expression of these markers during the early stages of CF lung disease. Methods: Bronchoalveolar lavage fluid (BALF) was collected from 50 children with CF aged 1, 3 or 5 years. BALF cells were analyzed using flow cytometry. Expression levels of surface markers on AMs were expressed as median fluorescence intensities (MFI) or percentage of AMs positive for these markers. The effect of age and neutrophilic inflammation, among other variables, on marker expression was assessed with a multivariate linear regression model. Results: AM expression of scavenger receptor CD163 decreased with age (p = 0.016) and was negatively correlated with BALF %neutrophils (r = -0.34, p = 0.016). AM expression of immune checkpoint molecule SIRPα also decreased with age (p = 0.0006), but did not correlate with BALF %neutrophils. Percentage of AMs expressing lipid scavenger CD36 was low overall (mean 20.1% ± 16.5) and did not correlate with other factors. Conversely, expression of immune checkpoint PD-1 was observed on the majority of AMs (mean PD-1pos 72.9% ± 11.8), but it, too, was not affected by age or BALF %neutrophils. Compared to matched blood monocytes, AMs had a higher expression of CD16, CD91, and PD-1, and a lower expression of CD163, SIRPα and CD36. Conclusion: In BALF of preschool children with CF, higher age and/or increased neutrophilic inflammation coincided with decreased expression of scavenger receptors on AMs. Expression of scavenging receptors and regulators showed a distinctly different pattern in AMs compared to blood monocytes. These findings suggest AM capacity to counter inflammation and promote homeostasis reduces during initiation of CF airway disease and highlight new avenues of investigation into impaired CF AM function.

Substrate-dependent metabolomic signatures of myeloperoxidase activity in airway epithelial cells: Implications for early cystic fibrosis lung disease.

Myeloperoxidase (MPO) is released by neutrophils in inflamed tissues. MPO oxidizes chloride, bromide, and thiocyanate to produce hypochlorous acid (HOCl), hypobromous acid (HOBr), and hypothiocyanous acid (HOSCN), respectively. These oxidants are toxic to pathogens, but may also react with host cells to elicit biological activity and potential toxicity. In cystic fibrosis (CF) and related diseases, increased neutrophil inflammation leads to increased airway MPO and airway epithelial cell (AEC) exposure to its oxidants. In this study, we investigated how equal dose-rate exposures of MPO-derived oxidants differentially impact the metabolome of human AECs (BEAS-2B cells). We utilized enzymatic oxidant production with rate-limiting glucose oxidase (GOX) coupled to MPO, and chloride, bromide (Br-), or thiocyanate (SCN-) as substrates. AECs exposed to GOX/MPO/SCN- (favoring HOSCN) were viable after 24 h, while exposure to GOX/MPO (favoring HOCl) or GOX/MPO/Br- (favoring HOBr) developed cytotoxicity after 6 h. Cell glutathione and peroxiredoxin-3 oxidation were insufficient to explain these differences. However, untargeted metabolomics revealed GOX/MPO and GOX/MPO/Br- diverged significantly from GOX/MPO/SCN- for dozens of metabolites. We noted methionine sulfoxide and dehydromethionine were significantly increased in GOX/MPO- or GOX/MPO/Br--treated cells, and analyzed them as potential biomarkers of lung damage in bronchoalveolar lavage fluid from 5-year-olds with CF (n = 27). Both metabolites were associated with increasing bronchiectasis, neutrophils, and MPO activity. This suggests MPO production of HOCl and/or HOBr may contribute to inflammatory lung damage in early CF. In summary, our in vitro model enabled unbiased identification of exposure-specific metabolite products which may serve as biomarkers of lung damage in vivo. Continued research with this exposure model may yield additional oxidant-specific biomarkers and reveal explicit mechanisms of oxidant byproduct formation and cellular redox signaling.

Transcriptional reprogramming of infiltrating neutrophils drives lung pathology in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. However, targeted studies on this vulnerable population are scarce. Here, we applied multiomics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, leads to persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to acute respiratory distress syndrome in a well-defined patient population disproportionally affected by severe COVID-19.

Immunometabolic Analysis of Synovial Fluid from Juvenile Idiopathic Arthritis Patients.

Juvenile idiopathic arthritis (JIA) is an inflammatory rheumatic disorder. Polymorphonuclear neutrophils (PMNs) are present in JIA synovial fluid (SF), but with variable frequency. SF PMNs in JIA were previously shown to display high exocytic but low phagocytic and immunoregulatory activities. To further assess whether the degree of SF neutrophilia associated with altered immune responses in JIA, we collected SF and blood from 16 adolescent JIA patients. SF and blood leukocytes were analyzed by flow cytometry. SF and plasma were used for immune mediator quantification and metabolomics. Healthy donor blood T cells were cultured in SF to evaluate its immunoregulatory activities. PMN and T cell frequencies were bimodal in JIA SF, delineating PMN high/T cell low (PMNHigh) and PMN low/T cell high (PMNLow) samples. Proinflammatory mediators were increased in SF compared with plasma across patients, and pro- and anti-inflammatory mediators were further elevated in PMNHigh SF. Compared to blood, SF PMNs showed increased exocytosis and programmed death-1/programmed death ligand-1 expression, and SF PMNs and monocytes/macrophages had increased surface-bound arginase-1. SPADE analysis revealed SF monocyte/macrophage subpopulations coexpressing programmed death-1 and programmed death ligand-1, with higher expression in PMNHigh SF. Healthy donor T cells showed reduced coreceptor expression when stimulated in PMNHigh versus PMNLow SF. However, amino acid metabolites related to the arginase-1 and IDO-1 pathways did not differ between the two groups. Hence, PMN predominance in the SF of a subset of JIA patients is associated with elevated immune mediator concentration and may alter SF monocyte/macrophage phenotype and T cell activation, without altering immunoregulatory amino acids.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins.

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Inhibition of Recruitment and Activation of Neutrophils by Pyridazinone-Scaffold-Based Compounds.

In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing exacerbated cell responses and tissue damage, novel therapeutic agents are sought. 4aa and 4ba, two pyridazinone-scaffold-based phosphodiesterase-IV inhibitors are compared in vitro to zardaverine for their ability to: (1) modulate production of pro-inflammatory mediators, reactive oxygen species (ROS), and phagocytosis; (2) modulate degranulation by PMNs after transepithelial lung migration. Compound 4ba and zardaverine were tested in vivo for their ability to limit tissue recruitment of PMNs in a murine air pouch model. In vitro treatment of lipopolysaccharide-stimulated PMNs with compounds 4aa and 4ba inhibited the release of interleukin-8, tumor necrosis factor-α, and matrix metalloproteinase-9. PMNs phagocytic ability, but not ROS production, was reduced following treatment. Using a lung inflammation model, we proved that PMNs transmigration led to reduced expression of the CD16 phagocytic receptor, which was significantly blunted after treatment with compound 4ba or zardaverine. Using the murine air pouch model, LPS-induced PMNs recruitment was significantly decreased upon addition of compound 4ba or zardaverine. Our data suggest that new pyridazinone derivatives have therapeutic potential in inflammatory diseases by limiting tissue recruitment and activation of PMNs.

Baricitinib attenuates the proinflammatory phase of COVID-19 driven by lung-infiltrating monocytes.

SARS-CoV-2-infected subjects are generally asymptomatic during initial viral replication but may suffer severe immunopathology after the virus has receded and monocytes have infiltrated the airways. In bronchoalveolar lavage fluid from severe COVID-19 patients, monocytes express mRNA encoding inflammatory mediators and contain SARS-CoV-2 transcripts. We leverage a human small airway model of infection and inflammation, whereby primary blood monocytes transmigrate across SARS-CoV-2-infected lung epithelium to characterize viral burden, gene expression, and inflammatory mediator secretion by epithelial cells and monocytes. In this model, lung-infiltrating monocytes acquire SARS-CoV-2 from the epithelium and upregulate expression and secretion of inflammatory mediators, mirroring in vivo data. Combined use of baricitinib (Janus kinase inhibitor) and remdesivir (nucleoside analog) enhances antiviral signaling and viral clearance by SARS-CoV-2-positive monocytes while decreasing secretion of proneutrophilic mediators associated with acute respiratory distress syndrome. These findings highlight the role of lung-infiltrating monocytes in COVID-19 pathogenesis and their importance as a therapeutic target.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease.

BACKGROUND: Macrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF. METHODS: Blood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays. RESULTS: Airway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance. CONCLUSION: Taken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.

Pilot study of inflammatory biomarkers in matched induced sputum and bronchoalveolar lavage of 2-year-olds with cystic fibrosis.

BACKGROUND: In this pilot study, we investigated whether induced sputum (IS) could serve as a viable alternative to bronchoalveolar lavage (BAL) and yield robust inflammatory biomarkers in toddlers with cystic fibrosis (CF) featuring minimal structural lung disease. METHODS: We collected IS, BAL (right middle lobe and lingula), and blood, and performed chest computed tomography (CT) scans from 2-year-olds with CF (N = 11), all within a single visit. Inflammatory biomarkers included 20 soluble immune mediators and neutrophil elastase (NE), as well as frequency and phenotype of T cells, monocytes/macrophages, and neutrophils. RESULTS: At the molecular level, nine mediators showed similar levels in IS and BAL (CXCL1, CXCL8, IL-1α, IL-1RA, IL-6, CCL2, CXCL10, M-CSF, VEGF-A), four were higher in IS than in BAL (CXCL5, IL-1ß, CXCL11, TNFSF10), and two were present in IS, but undetectable in BAL (IL-10, IFN-γ). Meanwhile, soluble NE had lower activity in IS than in BAL. At the cellular level, T-cell frequency was lower in IS than in BAL. Monocytes/macrophages were dominant in IS and BAL with similar frequencies, but differing expression of CD16 (lower in IS), CD115, and surface-associated NE (higher in IS). Neutrophil frequency and phenotype did not differ between IS and BAL. Finally, neutrophil frequency in IS correlated positively with air trapping. CONCLUSIONS: IS collected from 2-year-olds with CF yields biomarkers of early airway inflammation with good agreement with BAL, notably with regard to molecular and cellular outcomes related to neutrophils and monocytes/macrophages.

Sex differences in the relationships between body composition, fat distribution, and mitochondrial energy metabolism: a pilot study.

BACKGROUND: Adiposity and mitochondrial dysfunction are related factors contributing to metabolic disease development. This pilot study examined whether in vivo and ex vivo indices of mitochondrial metabolism were differentially associated with body composition in males and females. METHODS: Thirty-four participants including 19 females (mean 27 yr) and 15 males (mean 29 yr) had body composition assessed by dual energy x-ray absorptiometry and magnetic resonance (MR) imaging. Monocyte reserve capacity and maximal oxygen consumption rate (OCR) were determined ex vivo using extracellular flux analysis. In vivo quadriceps mitochondrial function was measured using 31P-MR spectroscopy based on post-exercise recovery kinetics (τPCr). The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin levels. Variables were log-transformed, and Pearson correlations and partial correlations were used for analyses. RESULTS: Mitochondrial metabolism was similar between sexes (p > 0.05). In males only, higher fat mass percent (FM%) was correlated with lower reserve capacity (r = - 0.73; p = 0.002) and reduced muscle mitochondrial function (r = 0.58, p = 0.02). Thigh subcutaneous adipose tissue was inversely related to reserve capacity in males (r = - 0.75, p = 0.001), but in females was correlated to higher maximal OCR (r = 0.48, p = 0.046), independent of FM. In females, lean mass was related to greater reserve capacity (r = 0.47, p = 0.04). In all participants, insulin (r = 0.35; p = 0.04) and HOMA-IR (r = 0.34; p = 0.05) were associated with a higher τPCr. CONCLUSIONS: These novel findings demonstrate distinct sex-dependent associations between monocyte and skeletal muscle mitochondrial metabolism with body composition. With further study, increased understanding of these relationships may inform sex-specific interventions to improve mitochondrial function and metabolic health.

Pseudomonas aeruginosa modulates neutrophil granule exocytosis in an in vitro model of airway infection.

A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air-liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP-10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin-1α and ß were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral-bacterial co-infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV-infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N-formylmethionyl-leucyl-phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.

Neutrophil-derived extracellular vesicles promote feed-forward inflammasome signaling in cystic fibrosis airways.

Cystic fibrosis (CF) airways feature high extracellular levels of the IL-1 family of proinflammatory mediators. These mediators are cleavage products of caspase-1, the final protease in the inflammasome cascade. Due to the proven chronic presence of reprogrammed neutrophils in the CF airway lumen, understanding inflammasome signaling in these cells is of great importance to understand how disease is perpetuated in this milieu. Here, we hypothesized that CF airway neutrophils contribute to chronic inflammation, in part, via the packaging of inflammasome-inducing signals in extracellular vesicles (EVs). We confirmed that CF airway fluid is enriched in IL-1α, IL-1ß, and IL-18, and that CF airway neutrophils up-regulate the activating receptor IL-1R1. Meanwhile, down-modulatory signals such as IL-1R2 and IL-1RA are unchanged. Active caspase-1 itself is present in CF airway fluid EVs, with neutrophil-derived EVs being most enriched. Using a transmigration model of CF airway inflammation, we show that CF airway fluid EVs are necessary and sufficient to induce primary granule exocytosis by naïve neutrophils (hallmark of reprogramming) and concomitantly activate caspase-1 and IL-1ß production by these cells and that the addition of triple-combination highly effective CFTR modulator therapy does not abrogate these effects. Finally, EVs from activated neutrophils can deliver active caspase-1 to primary tracheal epithelial cells and induce their release of IL-1α. These findings support the existence of a feed-forward inflammatory process by which reprogrammed CF airway neutrophils bypass 2-step control of inflammasome activation in neighboring cells (naïve neutrophils and epithelial cells) via the transfer of bioactive EVs.