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A rapid, quantitative serum S100A8/A9 (calprotectin) lateral flow test in combination with clinical status predicted outcomes in people hospitalised with COVID-19 and associated with a patient cluster driven by markers of neutrophil activation https://bit.ly/48e1BIv., Competing Interests: Conflict of interest: A.A.R. Thompson reports grants or contracts from British Heart Foundation, Heart Research UK, and National Institute for Health and Care Research, outside the submitted work; honoraria for lectures from Janssen-Cilag Ltd, outside the submitted work; and support received for travel and conference registration from Janssen-Cilag Ltd, outside the submitted work. Conflict of interest: A.M. Condliffe reports grants or contracts from NIHR and MRC, outside the submitted work. Conflict of interest: A. Shoemark reports grants or contracts from AstraZeneca, outside the submitted work; consulting fees from Spirovant and Translate Bio, outside the submitted work; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events for Translate Bio, Ethris and Insmed, outside the submitted work; and involvement in European Respiratory Society Clinical Research Collaborations (EMBARC, BEATPCD and AMR), EMBARC3 is supported by project partners Armata, AstraZeneca, Boehringer Ingelheim, Chiesi, CSL Behring, Grifols, Insmed, Janssen, Lifearc, Novartis, and Zambon, disclosures made outside the submitted work. Conflict of interest: J.D. Chalmers reports grants or contracts from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Gilead Sciences, Grifols, Novartis, Insmed and Trudell, outside the submitted work; and consulting fees from Antabio, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Insmed, Janssen, Novartis, Pfizer, Trudell and Zambon, outside the submitted work; and is an associate editor of this journal. Conflict of interest: The remaining authors have nothing to disclose., (Copyright ©The authors 2024.)

Rationale: Assessing the early use of video-assisted thoracoscopic surgery (VATS) or intrapleural enzyme therapy (IET) in pleural infection requires a phase III randomized controlled trial (RCT). Objectives: To establish the feasibility of randomization in a surgery-versus-nonsurgery trial as well as the key outcome measures that are important to identify relevant patient-centered outcomes in a subsequent RCT. Methods: The MIST-3 (third Multicenter Intrapleural Sepsis Trial) was a prospective multicenter RCT involving eight U.K. centers combining on-site and off-site surgical services. The study enrolled all patients with a confirmed diagnosis of pleural infection and randomized those with ongoing pleural sepsis after an initial period (as long as 24 h) of standard care to one of three treatment arms: continued standard care, early IET, or a surgical opinion with regard to early VATS. The primary outcome was feasibility based on >50% of eligible patients being successfully randomized, >95% of randomized participants retained to discharge, and >80% of randomized participants retained to 2 weeks of follow-up. The analysis was performed per intention to treat. Measurements and Main Results: Of 97 eligible patients, 60 (62%) were randomized, with 100% retained to discharge and 84% retained to 2 weeks. Baseline demographic, clinical, and microbiological characteristics of the patients were similar across groups. Median times to intervention were 1.0 and 3.5 days in the IET and surgery groups, respectively ( P  = 0.02). Despite the difference in time to intervention, length of stay (from randomization to discharge) was similar in both intervention arms (7 d) compared with standard care (10 d) ( P  = 0.70). There were no significant intergroup differences in 2-month readmission and further intervention, although the study was not adequately powered for this outcome. Compared with VATS, IET demonstrated a larger improvement in mean EuroQol five-dimension health utility index (five-level edition) from baseline (0.35) to 2 months (0.83) ( P  = 0.023). One serious adverse event was reported in the VATS arm. Conclusions: This is the first multicenter RCT of early IET versus early surgery in pleural infection. Despite the logistical challenges posed by the coronavirus disease (COVID-19) pandemic, the study met its predefined feasibility criteria, demonstrated potential shortening of length of stay with early surgery, and signals toward earlier resolution of pain and a shortened recovery with IET. The study findings suggest that a definitive phase III study is feasible but highlights important considerations and significant modifications to the design that would be required to adequately assess optimal initial management in pleural infection.The trial was registered on ISRCTN (number 18,192,121).

Protracted bacterial bronchitis (PBB) causes chronic wet cough for which seasonal azithromycin is increasingly used to reduce exacerbations. We investigated the impact of seasonal azithromycin on antimicrobial resistance and the nasopharyngeal microbiome. In an observational cohort study, 50 children with PBB were enrolled over two consecutive winters; 25/50 at study entry were designated on clinical grounds to take azithromycin over the winter months and 25/50 were not. Serial nasopharyngeal swabs were collected during the study period (12-20 months) and cultured bacterial isolates were assessed for antimicrobial susceptibility. 16S rRNA-based sequencing was performed on a subset of samples. Irrespective of azithromycin usage, high levels of azithromycin resistance were found; 73% of bacteria from swabs in the azithromycin group vs. 69% in the comparison group. Resistance was predominantly driven by azithromycin-resistant S. pneumoniae , yet these isolates were mostly erythromycin susceptible. Analysis of 16S rRNA-based sequencing revealed a reduction in within-sample diversity in response to azithromycin, but only in samples of children actively taking azithromycin at the time of swab collection. Actively taking azithromycin at the time of swab collection significantly contributed to dissimilarity in bacterial community composition. The discrepancy between laboratory detection of azithromycin and erythromycin resistance in the S. pneumoniae isolates requires further investigation. Seasonal azithromycin for PBB did not promote antimicrobial resistance over the study period, but did perturb the microbiome.

Background: Activated phosphoinositide-3-kinase δ syndrome (APDS) is an inborn error of immunity (IEI) with infection susceptibility and immune dysregulation, clinically overlapping with other conditions. Management depends on disease evolution, but predictors of severe disease are lacking., Objectives: This study sought to report the extended spectrum of disease manifestations in APDS1 versus APDS2; compare these to CTLA4 deficiency, NFKB1 deficiency, and STAT3 gain-of-function (GOF) disease; and identify predictors of severity in APDS., Methods: Data was collected from the ESID (European Society for Immunodeficiencies)-APDS registry and was compared with published cohorts of the other IEIs., Results: The analysis of 170 patients with APDS outlines high penetrance and early onset of APDS compared to the other IEIs. The large clinical heterogeneity even in individuals with the same PIK3CD variant E1021K illustrates how poorly the genotype predicts the disease phenotype and course. The high clinical overlap between APDS and the other investigated IEIs suggests relevant pathophysiological convergence of the affected pathways. Preferentially affected organ systems indicate specific pathophysiology: bronchiectasis is typical of APDS1; interstitial lung disease and enteropathy are more common in STAT3 GOF and CTLA4 deficiency. Endocrinopathies are most frequent in STAT3 GOF, but growth impairment is also common, particularly in APDS2. Early clinical presentation is a risk factor for severe disease in APDS., Conclusions: APDS illustrates how a single genetic variant can result in a diverse autoimmune-lymphoproliferative phenotype. Overlap with other IEIs is substantial. Some specific features distinguish APDS1 from APDS2. Early onset is a risk factor for severe disease course calling for specific treatment studies in younger patients., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Rationale: Sonographic septations are assumed to be important clinical predictors of outcome in pleural infection, but the evidence for this is sparse. The inflammatory and fibrinolysis-associated intrapleural pathway(s) leading to septation formation have not been studied in a large cohort of pleural fluid (PF) samples with confirmed pleural infection matched with ultrasound and clinical outcome data. Objectives: To assess the presence and severity of septations against baseline PF PAI-1 (Plasminogen-Activator Inhibitor-1) and other inflammatory and fibrinolysis-associated proteins as well as to correlate these with clinically important outcomes. Methods: We analyzed 214 pleural fluid samples from PILOT (Pleural Infection Longitudinal Outcome Study), a prospective observational pleural infection study, for inflammatory and fibrinolysis-associated proteins using the Luminex platform. Multivariate regression analyses were used to assess the association of pleural biological markers with septation presence and severity (on ultrasound) and clinical outcomes. Measurements and Main Results: PF PAI-1 was the only protein independently associated with septation presence ( P  < 0.001) and septation severity ( P  = 0.003). PF PAI-1 concentrations were associated with increased length of stay ( P  = 0.048) and increased 12-month mortality ( P  = 0.003). Sonographic septations alone had no relation to clinical outcomes. Conclusions: In a large and well-characterized cohort, this is the first study to associate pleural biological parameters with a validated sonographic septation outcome in pleural infection. PF PAI-1 is the first biomarker to demonstrate an independent association with mortality. Although PF PAI-1 plays an integral role in driving septation formation, septations themselves are not associated with clinically important outcomes. These novel findings now require prospective validation.

People with activated PI3 kinase delta syndrome 1 (APDS1) suffer from immune deficiency and severe bronchiectasis. APDS1 is caused by dominant activating mutations of the PIK3CD gene that encodes the PI3 kinase delta (PI3Kδ) catalytic subunit. Despite the importance of innate immunity defects in bronchiectasis, there has been limited investigation of neutrophils or macrophages in APDS1 patients or mouse models. Zebrafish embryos provide an ideal system to study neutrophils and macrophages. We used CRISPR-Cas9 and CRISPR-Cpf1, with oligonucleotide-directed homologous repair, to engineer zebrafish equivalents of the two most prevalent human APDS1 disease mutations. These zebrafish pik3cd alleles dominantly caused excessive neutrophilic inflammation in a tail-fin injury model. They also resulted in total body neutrophilia in the absence of any inflammatory stimulus but normal numbers of macrophages. Exposure of zebrafish to the PI3Kδ inhibitor CAL-101 reversed the total body neutrophilia. There was no apparent defect in neutrophil maturation or migration, and tail-fin regeneration was unimpaired. Overall, the finding is of enhanced granulopoeisis, in the absence of notable phenotypic change in neutrophils and macrophages., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Bacterial pathogens are confronted with a range of challenges at the site of infection, including exposure to antibiotic treatment and harsh physiological conditions, that can alter the fitness benefits and costs of acquiring antibiotic resistance. Here, we develop an experimental system to recapitulate resistance gene acquisition by Staphylococcus aureus and test how the subsequent evolution of the resistant bacterium is modulated by antibiotic treatment and oxygen levels, both of which are known to vary extensively at sites of infection. We show that acquiring tetracycline resistance was costly, reducing competitive growth against the isogenic strain without the resistance gene in the absence of the antibiotic, for S. aureus under hypoxic but not normoxic conditions. Treatment with tetracycline or doxycycline drove the emergence of enhanced resistance through mutations in an RluD-like protein-encoding gene and duplications of tetL , encoding the acquired tetracycline-specific efflux pump. In contrast, evolutionary adaptation by S. aureus to hypoxic conditions, which evolved in the absence of antibiotics through mutations affecting gyrB , was impeded by antibiotic treatment. Together, these data suggest that the horizontal acquisition of a new resistance mechanism is merely a starting point for the emergence of high-level resistance under antibiotic selection but that antibiotic treatment constrains pathogen adaptation to other important environmental selective forces such as hypoxia, which in turn could limit the survival of these highly resistant but poorly adapted genotypes after antibiotic treatment is ended.

Background: Neutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19., Methods: In a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012., Findings: Between June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57-0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug., Interpretation: Brensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19., Funding: Sponsored by the University of Dundee and supported through an Investigator Initiated Research award from Insmed, Bridgewater, NJ; STOP-COVID19 trial., Competing Interests: Declaration of interests JDC reports grants and personal fees from AstraZeneca, Boehringer-Ingelheim, Chiesi, GSK, Gilead Sciences, Grifols, Insmed, Janssen, Novartis, and Zambon. CB reports grants from the UK National Institute for Health and Care Research Biomedical Research Centre during the conduct of the study; grants and personal fees from GSK, AstraZeneca, Chiesi, Boehringer-Ingelheim, Genentech, Roche, Sanofi, Regeneron, Merck, TEVA, Mologic, 4DPharma, and Novartis. AART reports grants and personal fees from British Heart Foundation and Actelion Pharmaceuticals. JU reports personal fees from Gilead Sciences and ViiV Healthcare and from Celltrion; and is supported by the UK Medical Research Council (MR/T023791/1). DPSD reports grants and personal fees from GSK, Vir Biotechnology, AstraZeneca, and Boehringer-Ingelheim. ASm has received non-financial support for clinical trial work from AstraZeneca, GSK, Chiesi, and Oncimmune; and has done consultancy work with AstraZeneca and GSK. MP reports non-financial support for clinical trial work from AstraZeneca, GSK, Chiesi, and Oncimmune and consultancy work with AstraZeneca and GSK. All other authors report no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Immunocompromised individuals are at high risk of developing severe fungal infections with high mortality rates, while fungal pathogens pose little risk to most healthy people. Poor therapeutic outcomes and growing antifungal resistance pose further challenges for treatments. Identifying specific immunomodulatory mechanisms exploited by fungal pathogens is critical for our understanding of fungal diseases and development of new therapies. A gap currently exists between the large body of literature concerning the innate immune response to fungal infections and the potential manipulation of host immune responses to aid clearance of infection. This review considers the innate immune mechanisms the host deploys to prevent fungal infection and how these mechanisms fail in immunocompromised hosts. Three clinically relevant fungal pathogens ( Candida albicans , Cryptococcus spp. and Aspergillus spp.) will be explored. This review will also examine potential mechanisms of targeting the host therapeutically to improve outcomes of fungal infection.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Background: Neutrophil extracellular traps (NETs) are web-like DNA and protein lattices which are expelled by neutrophils to trap and kill pathogens, but which cause significant damage to the host tissue. NETs have emerged as critical mediators of lung damage, inflammation and thrombosis in coronavirus disease 2019 (COVID-19) and other diseases, but there are no therapeutics to prevent or reduce NETs that are available to patients., Methods: Neutrophils were isolated from healthy volunteers (n=9) and hospitalised patients with COVID-19 at the acute stage (n=39) and again at 3-4 months post-acute sampling (n=7). NETosis was measured by SYTOX green assays., Results: Here, we show that neutrophils isolated from hospitalised patients with COVID-19 produce significantly more NETs in response to lipopolysaccharide (LPS) compared to cells from healthy control subjects. A subset of patients was captured at follow-up clinics (3-4 months post-acute sampling), and while LPS-induced NET formation is significantly lower at this time point, it remains elevated compared to healthy controls. LPS- and phorbol myristate acetate (PMA)-induced NETs were significantly inhibited by the protein kinase C (PKC) inhibitor ruboxistaurin. Ruboxistaurin-mediated inhibition of NETs in healthy neutrophils reduces NET-induced epithelial cell death., Conclusion: Our findings suggest ruboxistaurin could reduce proinflammatory and tissue-damaging consequences of neutrophils during disease, and since it has completed phase III trials for other indications without safety concerns, it is a promising and novel therapeutic strategy for COVID-19., Competing Interests: Conflict of interest: R. Dowey has nothing to disclose. Conflict of interest: J. Cole has nothing to disclose. Conflict of interest: A.A.R. Thompson has nothing to disclose. Conflict of interest: R.C. Hull has nothing to disclose. Conflict of interest: C. Huang has nothing to disclose. Conflict of interest: J. Whatmore has nothing to disclose. Conflict of interest: A. Iqbal has nothing to disclose. Conflict of interest: K.L. Bradley has nothing to disclose. Conflict of interest: J. McKenzie has nothing to disclose. Conflict of interest: A. Lawrie has nothing to disclose. Conflict of interest: A.M. Condliffe has nothing to disclose. Conflict of interest: E. Kiss-Toth has nothing to disclose. Conflict of interest: I. Sabroe has nothing to disclose. Conflict of interest: .R. Prince has nothing to disclose., (Copyright ©The authors 2022.)

Background: Hyperpolarised gas magnetic resonance imaging (MRI) can be used to assess ventilation patterns. Previous studies have shown the image-derived metric of ventilation defect per cent (VDP) to correlate with forced expiratory volume in 1 s (FEV 1 )/forced vital capacity (FVC) and FEV 1 in asthma., Objectives: The aim of this study was to explore the utility of hyperpolarised xenon-129 ( 129 Xe) ventilation MRI in clinical care and examine its relationship with spirometry and other clinical metrics in people seen in a severe asthma service., Methods: 26 people referred from a severe asthma clinic for MRI scanning were assessed by contemporaneous 129 Xe MRI and spirometry. A subgroup of 18 patients also underwent reversibility testing with spirometry and MRI. Quantitative MRI measures of ventilation were calculated, VDP and the ventilation heterogeneity index (VH I ), and compared to spirometry, Asthma Control Questionnaire 7 (ACQ7) and blood eosinophil count. Images were reviewed by a multidisciplinary team., Results: VDP and VH I correlated with FEV 1 , FEV 1 /FVC and forced expiratory flow between 25% and 75% of FVC but not with ACQ7 or blood eosinophil count. Discordance of MRI imaging and symptoms and/or pulmonary function tests also occurred, prompting diagnostic re-evaluation in some cases., Conclusion: Hyperpolarised gas MRI provides a complementary method of assessment in people with difficult to manage asthma in a clinical setting. When used as a tool supporting clinical care in a severe asthma service, occurrences of discordance between symptoms, spirometry and MRI scanning indicate how MRI scanning may add to a management pathway., Competing Interests: Conflict of interest: AstraZeneca awarded a research grant to I. Sabroe, J.M. Wild, H. Marshall and colleagues for the period 2020–2023 to study effects of therapies on airway imaging. In 2019, I. Sabroe participated in a scientific advisory board for AstraZeneca, generating a small consultancy income paid to the University of Sheffield to support research, and has attended an annual respiratory research meeting supported by Boehringer Ingelheim. J.M. Wild and H. Marshall attended and advised on an AstraZeneca global respiratory experts meeting, generating a small consultancy income paid to the University. All other authors declare no conflicts of interest related to this study., (Copyright ©The authors 2021.)

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives: To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice ( N  = 12) resulted in increased lung vascular permeability ( P  = 0.022), interstitial edema ( P  = 0.0047), and neutrophil extravasation ( P  = 0.029) compared with IgG control treatment ( N  = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice ( N  = 8) prevented inhaled LPS-induced lung injury ( P  = 0.0027) and edema ( P  < 0.0001). In endotoxemic mice ( N  = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis ( N  = 10), circulating concentratons of BMP9 were also markedly reduced ( P  < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease., Competing Interests: The authors have declared that no competing interests exist.

The lungs are exposed to a range of environmental toxins (including cigarette smoke, air pollution, asbestos) and pathogens (bacterial, viral and fungal), and most respiratory diseases are associated with local or systemic hypoxia. All of these adverse factors can trigger endoplasmic reticulum (ER) stress. The ER is a key intracellular site for synthesis of secretory and membrane proteins, regulating their folding, assembly into complexes, transport and degradation. Accumulation of misfolded proteins within the lumen results in ER stress, which activates the unfolded protein response (UPR). Effectors of the UPR temporarily reduce protein synthesis, while enhancing degradation of misfolded proteins and increasing the folding capacity of the ER. If successful, homeostasis is restored and protein synthesis resumes, but if ER stress persists, cell death pathways are activated. ER stress and the resulting UPR occur in a range of pulmonary insults and the outcome plays an important role in many respiratory diseases. The UPR is triggered in the airway of patients with several respiratory diseases and in corresponding experimental models. ER stress has been implicated in the initiation and progression of pulmonary fibrosis, and evidence is accumulating suggesting that ER stress occurs in obstructive lung diseases (particularly in asthma), in pulmonary infections (some viral infections and in the setting of the cystic fibrosis airway) and in lung cancer. While a number of small molecule inhibitors have been used to interrogate the role of the UPR in disease models, many of these tools have complex and off-target effects, hence additional evidence (eg, from genetic manipulation) may be required to support conclusions based on the impact of such pharmacological agents. Aberrant activation of the UPR may be linked to disease pathogenesis and progression, but at present, our understanding of the context-specific and disease-specific mechanisms linking these processes is incomplete. Despite this, the ability of the UPR to defend against ER stress and influence a range of respiratory diseases is becoming increasingly evident, and the UPR is therefore attracting attention as a prospective target for therapeutic intervention strategies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Activated phosphoinositide 3-kinase delta syndrome (APDS) is a novel primary immunodeficiency (PID) caused by heterozygous gain of function mutations in PI3Kδ catalytic p110δ (PIK3CD) or regulatory p85α (PIK3R1) subunits leading to APDS1 and APDS2, respectively. Patients with APDS present a spectrum of clinical manifestations, particularly recurrent respiratory infections and lymphoproliferation. We searched PubMed, Web of Science, and Scopus databases for APDS patients and screened for eligibility criteria. A total of 243 APDS patients were identified from 55 articles. For all patients, demographic, clinical, immunologic, and molecular data were collected. Overall, 179 APDS1 and 64 APDS2 patients were identified. The most common clinical manifestations were respiratory tract infections (pneumonia (43.6%), otitis media (28.8%), and sinusitis (25.9%)), lymphoproliferation (70.4%), autoimmunity (28%), enteropathy (26.7%), failure to thrive (20.6%), and malignancy (12.8%). The predominant immunologic phenotype was hyper-IgM syndrome (48.1%). Immunologic profiling showed decreased B cells in 74.8% and CD4 + T cells in 64.8% of APDS patients. The c.3061 G>A (p. E1021K) mutation in APDS1 with 85% frequency and c.1425+1 G> (A, C, T) (p.434-475del) mutation in APDS2 with 79% frequency were hotspot mutations. The majority of APDS patients were placed on long-term immunoglobulin replacement therapy. Immunosuppressive agents such as rituximab, tacrolimus, rapamycin, and leniolisib were also administered for autoimmunity and inflammatory complications. In addition, hematopoietic stem cell transplantation (HSCT) was used in 12.8% of patients. APDS has heterogynous clinical manifestations. It should be suspected in patients with history of recurrent respiratory infections, lymphoproliferation, and raised IgM levels. Moreover, HSCT should be considered in patients with severe and complicated clinical manifestations with no or insufficient response to the conventional therapies.

Background: Granulomatous-lymphocytic interstitial lung disease (GLILD) is a rare, potentially severe pulmonary complication of common variable immunodeficiency disorders (CVID). Informative clinical trials and consensus on management are lacking., Aims: The European GLILD network (e-GLILDnet) aims to describe how GLILD is currently managed in clinical practice and to determine the main uncertainties and unmet needs regarding diagnosis, treatment and follow-up., Methods: The e-GLILDnet collaborators developed and conducted an online survey facilitated by the European Society for Immunodeficiencies (ESID) and the European Respiratory Society (ERS) between February-April 2020. Results were analyzed using SPSS., Results: One hundred and sixty-one responses from adult and pediatric pulmonologists and immunologists from 47 countries were analyzed. Respondents treated a median of 27 (interquartile range, IQR 82-maximum 500) CVID patients, of which a median of 5 (IQR 8-max 200) had GLILD. Most respondents experienced difficulties in establishing the diagnosis of GLILD and only 31 (19%) had access to a standardized protocol. There was little uniformity in diagnostic or therapeutic interventions. Fewer than 40% of respondents saw a definite need for biopsy in all cases or performed bronchoalveolar lavage for diagnostics. Sixty-six percent used glucocorticosteroids for remission-induction and 47% for maintenance therapy; azathioprine, rituximab and mycophenolate mofetil were the most frequently prescribed steroid-sparing agents. Pulmonary function tests were the preferred modality for monitoring patients during follow-up., Conclusions: These data demonstrate an urgent need for clinical studies to provide more evidence for an international consensus regarding management of GLILD. These studies will need to address optimal procedures for definite diagnosis and a better understanding of the pathogenesis of GLILD in order to provide individualized treatment options. Non-availability of well-established standardized protocols risks endangering patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 van de Ven, Alfaro, Robinson, Baumann, Bergeron, Burns, Condliffe, Fevang, Gennery, Haerynck, Jacob, Jolles, Malphettes, Meignin, Milota, van Montfrans, Prasse, Quinti, Renzoni, Stolz, Warnatz and Hurst.)

Klebsiella pneumoniae is a Gram-negative bacterium that is increasingly difficult to treat due to the emergence of multidrug resistant strains. In a recent article, Ding et al demonstrate that prekallikrein depletion in mice followed by intranasal instillation of K. pneumoniae leads to a reduced bacterial burden and prolonged host survival, together with evidence of reduced distant organ damage. These effects are apparently independent of the role of prekallikrein in the contact system, and are associated with transcriptional changes relevant to innate immunity in the lung, established prior to infection. This study highlights the importance of further investigating the role of prekallikrein and other contact cascade components in host defence to counter K. pneumoniae (and perhaps other pathogens), with an overall aim of identifying potential therapeutic targets relevant to pulmonary infection with such resistant pathogens. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Neutrophils are key effector cells of innate immunity, rapidly recruited to defend the host against invading pathogens. Neutrophils may kill pathogens intracellularly, following phagocytosis, or extracellularly, by degranulation and the release of neutrophil extracellular traps; all of these microbicidal strategies require the deployment of cytotoxic proteins and proteases, packaged during neutrophil development within cytoplasmic granules. Neutrophils operate in infected and inflamed tissues, which can be profoundly hypoxic. Neutrophilic infiltration of hypoxic tissues characterises a myriad of acute and chronic infectious and inflammatory diseases, and as well as potentially protecting the host from pathogens, neutrophil granule products have been implicated in causing collateral tissue damage in these scenarios. This review discusses the evidence for the enhanced secretion of destructive neutrophil granule contents observed in hypoxic environments and the potential mechanisms for this heightened granule exocytosis, highlighting implications for the host. Understanding the dichotomy of the beneficial and detrimental consequences of neutrophil degranulation in hypoxic environments is crucial to inform potential neutrophil-directed therapeutics in order to limit persistent, excessive, or inappropriate inflammation., Competing Interests: The authors declare no conflict of interest.

Neutrophils are key to host defence, and impaired neutrophil function predisposes to infection with an array of pathogens, with Staphylococcus aureus a common and sometimes life-threatening problem in this setting. Both infiltrating immune cells and replicating bacteria consume oxygen, contributing to the profound tissue hypoxia that characterises sites of infection. Hypoxia in turn has a dramatic effect on both neutrophil bactericidal function and the properties of S. aureus , including the production of virulence factors. Hypoxia thereby shapes the host-pathogen interaction and the progression of infection, for example promoting intracellular bacterial persistence, enabling local tissue destruction with the formation of an encaging abscess capsule, and facilitating the establishment and propagation of bacterial biofilms which block the access of host immune cells. Elucidating the molecular mechanisms underlying host-pathogen interactions in the setting of hypoxia will enable better understanding of persistent and recalcitrant infections due to S. aureus and may uncover novel therapeutic targets and strategies.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1 -/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1 -/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1 -/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.