Early mucosal events promote distinct mucosal and systemic antibody responses to live attenuated influenza vaccine.

Compared to intramuscular vaccines, nasally administered vaccines have the advantage of inducing local mucosal immune responses that may block infection and interrupt transmission of respiratory pathogens. Live attenuated influenza vaccine (LAIV) is effective in preventing influenza in children, but a correlate of protection for LAIV remains unclear. Studying young adult volunteers, we observe that LAIV induces distinct, compartmentalized, antibody responses in the mucosa and blood. Seeking immunologic correlates of these distinct antibody responses we find associations with mucosal IL-33 release in the first 8 hours post-inoculation and divergent CD8+ and circulating T follicular helper (cTfh) T cell responses 7 days post-inoculation. Mucosal antibodies are induced separately from blood antibodies, are associated with distinct immune responses early post-inoculation, and may provide a correlate of protection for mucosal vaccination. This study was registered as NCT04110366 and reports primary (mucosal antibody) and secondary (blood antibody, and nasal viral load and cytokine) endpoint data.

Nasal IL-13 production identifies patients with late-phase allergic responses.

BACKGROUND: There is limited knowledge on how local cytokine secretion patterns after nasal allergen challenge correlate with clinical symptoms especially with regard to the "late allergic response," which occurs in approximately 40% to 50% of patients with allergy. OBJECTIVE: We sought to characterize the immunologic and clinical nasal responses to birch pollen allergen challenge with a special focus on the late allergic response. METHODS: In this randomized, double-blind, placebo-controlled trial, birch pollen-allergic participants were challenged with birch pollen extract (n = 20) or placebo (n = 10) on 3 consecutive days. On days 1 and 3, nasal secretions were collected at selected time points over a 24-hour time course for the measurement of 33 inflammatory mediators. Clinical responses were determined through subjective symptom scores and objective nasal airflow measurements. RESULTS: Provoked participants had significantly greater clinical responses and showed significant increases in tryptase and the soluble IL-33 receptor serum stimulation 2 (sST2) in nasal secretions within minutes compared with the placebo group. Eight of 20 provoked participants displayed high IL-13 levels 2 to 8 hours after allergen provocation. This group also showed significant changes in clinical parameters, with a secondary drop in nasal airflow measured by peak nasal inspiratory flow and increased symptoms of nasal obstruction, which significantly differed from IL-13 nonresponders after 6 hours. CONCLUSIONS: IL-13 response status correlates with clinical responses and type 2 cytokine responses in the late phase after allergen provocation.

Cellular and molecular features of COVID-19 associated ARDS: therapeutic relevance.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection can be asymptomatic or cause a disease (COVID-19) characterized by different levels of severity. The main cause of severe COVID-19 and death is represented by acute (or acute on chronic) respiratory failure and acute respiratory distress syndrome (ARDS), often requiring hospital admission and ventilator support.The molecular pathogenesis of COVID-19-related ARDS (by now termed c-ARDS) is still poorly understood. In this review we will discuss the genetic susceptibility to COVID-19, the pathogenesis and the local and systemic biomarkers correlated with c-ARDS and the therapeutic options that target the cell signalling pathways of c-ARDS.

Pulmonary Innate Lymphoid Cell Responses during Rhinovirus-induced Asthma Exacerbations In Vivo: A Clinical Trial.

Rationale: Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well characterized. Objectives: To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. Methods: Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16 and underwent bronchoscopy at baseline and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. Measurements and Main Results: At baseline, ILC2s were significantly higher in patients with asthma than in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with asthma than in healthy subjects (all comparisons P < 0.05). In healthy subjects, ILC1s increased from baseline at Day 3 (P = 0.001), while in patients with asthma, ILC1s increased from baseline at Day 8 (P = 0.042). Patients with asthma had significantly higher ILC2:ILC1 ratios at baseline (P = 0.024) and Day 8 (P = 0.005). Increased ILC2:ILC1 ratio in patients with asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid. Conclusions: An ILC2-predominant inflammatory profile in patients with asthma was associated with increased severity and duration of rhinovirus infection compared with healthy subjects, supporting the potential role of ILC2s in the pathogenesis of virus-induced asthma exacerbations.

Increased nasal mucosal interferon and CCL13 response to a TLR7/8 agonist in asthma and allergic rhinitis.

BACKGROUND: Acute respiratory viral infections are a major cause of respiratory morbidity and mortality, especially in patients with preexisting lung diseases such as asthma. Toll-like receptors are critical in the early detection of viruses and in activating innate immunity in the respiratory mucosa, but there is no reliable and convenient method by which respiratory mucosal innate immune responses can be measured. OBJECTIVE: We sought to assess in vivo immune responses to an innate stimulus and compare responsiveness between healthy volunteers and volunteers with allergy. METHODS: We administered the Toll-like receptor 7/8 agonist resiquimod (R848; a synthetic analogue of single-stranded RNA) or saline by nasal spray to healthy participants without allergy (n = 12), those with allergic rhinitis (n = 12), or those with allergic rhinitis with asthma (n = 11). Immune mediators in blood and nasal fluid and mucosal gene expression were monitored over time. RESULTS: R848 was well tolerated and significantly induced IFN-α2a, IFN-γ, proinflammatory cytokines (TNF-α, IL-2, IL-12p70), and chemokines (CXCL10, C-C motif chemokine ligand [CCL]2, CCL3, CCL4, and CCL13) in nasal mucosal fluid, without causing systemic immune activation. Participants with allergic rhinitis or allergic rhinitis with asthma had increased IFN-α2a, CCL3, and CCL13 responses relative to healthy participants; those with asthma had increased induction of IFN-stimulated genes DExD/H-box helicase 58, MX dynamin-like GTPase 1, and IFN-induced protein with tetratricopeptide repeats 3. CONCLUSIONS: Responses to nasal delivery of R848 enables simple assessment of mucosal innate responsiveness, revealing that patients with allergic disorders have an increased nasal mucosal IFN and chemokine response to the viral RNA analogue R848. This highlights that dysregulated innate immune responses of the nasal mucosa in allergic individuals may be important in determining the outcome of viral exposure.

Noninvasive and minimally invasive techniques for the diagnosis and management of allergic diseases.

Allergic diseases of the (upper and lower) airways, the skin and the gastrointestinal tract, are on the rise, resulting in impaired quality of life, decreased productivity, and increased healthcare costs. As allergic diseases are mostly tissue-specific, local sampling methods for respective biomarkers offer the potential for increased sensitivity and specificity. Additionally, local sampling using noninvasive or minimally invasive methods can be cost-effective and well tolerated, which may even be suitable for primary or home care sampling. Non- or minimally invasive local sampling and diagnostics may enable a more thorough endotyping, may help to avoid under- or overdiagnosis, and may provide the possibility to approach precision prevention, due to early diagnosis of these local diseases even before they get systemically manifested and detectable. At the same time, dried blood samples may help to facilitate minimal-invasive primary or home care sampling for classical systemic diagnostic approaches. This EAACI position paper contains a thorough review of the various technologies in allergy diagnosis available on the market, which analytes or biomarkers are employed, and which samples or matrices can be used. Based on this assessment, EAACI position is to drive these developments to efficiently identify allergy and possibly later also viral epidemics and take advantage of comprehensive knowledge to initiate preventions and treatments.

Neutrophilic inflammation in the respiratory mucosa predisposes to RSV infection.

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.

A Rare Mutation in SPLUNC1 Affects Bacterial Adherence and Invasion in Meningococcal Disease.

BACKGROUND: Neisseria meningitidis (Nm) is a nasopharyngeal commensal carried by healthy individuals. However, invasive infections occurs in a minority of individuals, with devastating consequences. There is evidence that common polymorphisms are associated with invasive meningococcal disease (IMD), but the contributions of rare variants other than those in the complement system have not been determined. METHODS: We identified familial cases of IMD in the UK meningococcal disease study and the European Union Life-Threatening Infectious Disease Study. Candidate genetic variants were identified by whole-exome sequencing of 2 patients with familial IMD. Candidate variants were further validated by in vitro assays. RESULTS: Exomes of 2 siblings with IMD identified a novel heterozygous missense mutation in BPIFA1/SPLUNC1. Sequencing of 186 other nonfamilial cases identified another unrelated IMD patient with the same mutation. SPLUNC1 is an innate immune defense protein expressed in the nasopharyngeal epithelia; however, its role in invasive infections is unknown. In vitro assays demonstrated that recombinant SPLUNC1 protein inhibits biofilm formation by Nm, and impedes Nm adhesion and invasion of human airway cells. The dominant negative mutant recombinant SPLUNC1 (p.G22E) showed reduced antibiofilm activity, increased meningococcal adhesion, and increased invasion of cells, compared with wild-type SPLUNC1. CONCLUSIONS: A mutation in SPLUNC1 affecting mucosal attachment, biofilm formation, and invasion of mucosal epithelial cells is a new genetic cause of meningococcal disease.

Patterns of systemic and local inflammation in patients with asthma hospitalised with influenza.

BACKGROUND: Patients with asthma are at risk of hospitalisation with influenza, but the reasons for this predisposition are unknown. STUDY SETTING: A prospective observational study of adults with PCR-confirmed influenza in 11 UK hospitals, measuring nasal, nasopharyngeal and systemic immune mediators and whole-blood gene expression. RESULTS: Of 133 admissions, 40 (30%) had previous asthma; these were more often female (70% versus 38.7%, OR 3.69, 95% CI 1.67-8.18; p=0.0012), required less mechanical ventilation (15% versus 37.6%, Chi-squared 6.78; p=0.0338) and had shorter hospital stays (mean 8.3 versus 15.3 days, p=0.0333) than those without. In patients without asthma, severe outcomes were more frequent in those given corticosteroids (OR 2.63, 95% CI 1.02-6.96; p=0.0466) or presenting >4 days after disease onset (OR 5.49, 95% CI 2.28-14.03; p=0.0002). Influenza vaccination in at-risk groups (including asthma) were lower than intended by national policy and the early use of antiviral medications were less than optimal. Mucosal immune responses were equivalent between groups. Those with asthma had higher serum interferon (IFN)-α, but lower serum tumour necrosis factor, interleukin (IL)-5, IL-6, CXCL8, CXCL9, IL-10, IL-17 and CCL2 levels (all p<0.05); both groups had similar serum IL-13, total IgE, periostin and blood eosinophil gene expression levels. Asthma diagnosis was unrelated to viral load, IFN-α, IFN-γ, IL-5 or IL-13 levels. CONCLUSIONS: Asthma is common in those hospitalised with influenza, but may not represent classical type 2-driven disease. Those admitted with influenza tend to be female with mild serum inflammatory responses, increased serum IFN-α levels and good clinical outcomes.

Biomarkers for Acute Respiratory Distress syndrome and prospects for personalised medicine.

Acute lung injury (ALI) affects over 10% of patients hospitalised in critical care, with acute respiratory distress syndrome (ARDS) being the most severe form of ALI and having a mortality rate in the region of 40%. There has been slow but incremental progress in identification of biomarkers that contribute to the pathophysiology of ARDS, have utility in diagnosis and monitoring, and that are potential therapeutic targets (Calfee CS, Delucchi K, Parsons PE, Thompson BT, Ware LB, Matthay MA, Thompson T, Ware LB, Matthay MA, Lancet Respir Med 2014, 2:611--620). However, a major issue is that ARDS is such a heterogeneous, multi-factorial, end-stage condition that the strategies for "lumping and splitting" are critical (Prescott HC, Calfee CS, Thompson BT, Angus DC, Liu VX, Am J Respir Crit Care Med 2016, 194:147--155). Nevertheless, sequencing of the human genome, the availability of improved methods for analysis of transcription to mRNA (gene expression), and development of sensitive immunoassays has allowed the application of network biology to ARDS, with these biomarkers offering potential for personalised or precision medicine (Sweeney TE, Khatri P, Toward precision medicine Crit Care Med; 2017 45:934-939). Biomarker panels have potential applications in molecular phenotyping for identifying patients at risk of developing ARDS, diagnosis of ARDS, risk stratification and monitoring. Two subphenotypes of ARDS have been identified on the basis of blood biomarkers: hypo-inflammatory and hyper-inflammatory. The hyper-inflammatory subphenotype is associated with shock, metabolic acidosis and worst clinical outcomes. Biomarkers of particular interest have included interleukins (IL-6 and IL-8), interferon gamma (IFN-γ), surfactant proteins (SPD and SPB), von Willebrand factor antigen, angiopoietin 1/2 and plasminogen activator inhibitor-1 (PAI-1). In terms of gene expression (mRNA) in blood there have been found to be increases in neutrophil-related genes in sepsis-induced and influenza-induced ARDS, but whole blood expression does not give a robust diagnostic test for ARDS. Despite improvements in management of ARDS on the critical care unit, this complex disease continues to be a major life-threatening event. Clinical trials of ß2-agonists, statins, surfactants and keratinocyte growth factor (KGF) have been disappointing. In addition, monoclonal antibodies (anti-TNF) and TNFR fusion protein have also been unconvincing. However, there have been major advances in methods of mechanical ventilation, a neuromuscular blocker (cisatracurium besilate) has shown some benefit, and stem cell therapy is being developed. In the future, by understanding the role of biomarkers in the pathophysiology of ARDS and lung injury, it is hoped that this will provide rational therapeutic targets and ultimately improve clinical care (Seymour CW, Gomez H, Chang CH, Clermont G, Kellum JA, Kennedy J, Yende S, Angus DC, Crit Care 2017, 21:257).

Reduced Nasal Viral Load and IFN Responses in Infants with Respiratory Syncytial Virus Bronchiolitis and Respiratory Failure.

RATIONALE: Respiratory syncytial virus (RSV) bronchiolitis is a major cause of morbidity and mortality in infancy. Severe disease is believed to result from uncontrolled viral replication, an excessive immune response, or both. OBJECTIVES: To determine RSV load and immune mediator levels in nasal mucosal lining fluid by serial sampling of nasal fluids from cases of moderate and severe bronchiolitis over the course of infection. METHODS: Infants with viral bronchiolitis necessitating admission (n = 55) were recruited from a pediatric center during 2016 and 2017. Of these, 30 were RSV infected (18 "moderate" and 12 mechanically ventilated "severe"). Nasal fluids were sampled frequently over time using nasosorption devices and nasopharyngeal aspiration. Hierarchical clustering of time-weighted averages was performed to investigate cytokine and chemokine levels, and gene expression profiling was conducted. MEASUREMENTS AND MAIN RESULTS: Unexpectedly, cases with severe RSV bronchiolitis had lower nasal viral loads and reduced IFN-γ and C-C chemokine ligand 5/RANTES (regulated upon activation, normal T cell expressed and secreted) levels than those with moderate disease, especially when allowance was made for disease duration (all P < 0.05). Reduced cytokine/chemokine levels in severe disease were also seen in children with other viral infections. Gene expression analysis of nasopharyngeal aspiration samples (n = 43) confirmed reduced type-I IFN gene expression in severe bronchiolitis accompanied by enhanced expression of MUC5AC and IL17A. CONCLUSIONS: Infants with severe RSV bronchiolitis have lower nasal viral load, CXCL10 (C-X-C motif chemokine ligand 10)/IP-10, and type-I IFN levels than moderately ill children, but enhanced MUC5AC (mucin-5AC) and IL17A gene expression in nasal cells.

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable. BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples. This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

A Comprehensive Evaluation of Nasal and Bronchial Cytokines and Chemokines Following Experimental Rhinovirus Infection in Allergic Asthma: Increased Interferons (IFN-γ and IFN-λ) and Type 2 Inflammation (IL-5 and IL-13).

BACKGROUND: Rhinovirus infection is a major cause of asthma exacerbations. OBJECTIVES: We studied nasal and bronchial mucosal inflammatory responses during experimental rhinovirus-induced asthma exacerbations. METHODS: We used nasosorption on days 0, 2-5 and 7 and bronchosorption at baseline and day 4 to sample mucosal lining fluid to investigate airway mucosal responses to rhinovirus infection in patients with allergic asthma (n=28) and healthy non-atopic controls (n=11), by using a synthetic absorptive matrix and measuring levels of 34 cytokines and chemokines using a sensitive multiplex assay. RESULTS: Following rhinovirus infection asthmatics developed more upper and lower respiratory symptoms and lower peak expiratory flows compared to controls (all P<0.05). Asthmatics also developed higher nasal lining fluid levels of an anti-viral pathway (including IFN-γ, IFN-λ/IL-29, CXCL11/ITAC, CXCL10/IP10 and IL-15) and a type 2 inflammatory pathway (IL-4, IL-5, IL-13, CCL17/TARC, CCL11/eotaxin, CCL26/eotaxin-3) (area under curve day 0-7, all P<0.05). Nasal IL-5 and IL-13 were higher in asthmatics at day 0 (P<0.01) and levels increased by days 3 and 4 (P<0.01). A hierarchical correlation matrix of 24 nasal lining fluid cytokine and chemokine levels over 7days demonstrated expression of distinct interferon-related and type 2 pathways in asthmatics. In asthmatics IFN-γ, CXCL10/IP10, CXCL11/ITAC, IL-15 and IL-5 increased in bronchial lining fluid following viral infection (all P<0.05). CONCLUSIONS: Precision sampling of mucosal lining fluid identifies robust interferon and type 2 responses in the upper and lower airways of asthmatics during an asthma exacerbation. Nasosorption and bronchosorption have potential to define asthma endotypes in stable disease and at exacerbation.

Nasosorption as a Minimally Invasive Sampling Procedure: Mucosal Viral Load and Inflammation in Primary RSV Bronchiolitis.

Background: Existing respiratory mucosal sampling methods are flawed, particularly in a pediatric bronchiolitis setting. Methods: Twenty-four infants with bronchiolitis were recruited: 12 were respiratory syncytial virus (RSV)-positive, 12 were RSV-negative. Infants were sampled by nasosorption and nasopharyngeal aspiration (NPA). Results: Nasosorption was well tolerated and identified all RSV+ samples. RSV load measured by nasosorption (but not NPA) correlated with length of hospital stay (P = .04) and requirement for mechanical ventilation (P = .03). Nasosorption (but not NPA) levels of interferon γ, interleukin 1ß, CCL5/RANTES, and interleukin 10 (IL-10) were elevated in RSV+ bronchiolitis (all P < .05), furthermore CCL5 and IL-10 correlated with RSV load (P < .05). Conclusions: Nasosorption allowed measurement of RSV load and the mucosal inflammatory response in infants.

Medical Swab Analysis Using Desorption Electrospray Ionization Mass Spectrometry: A Noninvasive Approach for Mucosal Diagnostics.

Medical swabs are routinely used worldwide to sample human mucosa for microbiological screening with culture methods. These are usually time-consuming and have a narrow focus on screening for particular microorganism species. As an alternative, direct mass spectrometric profiling of the mucosal metabolome provides a broader window into the mucosal ecosystem. We present for the first time a minimal effort/minimal-disruption technique for augmenting the information obtained from clinical swab analysis with mucosal metabolome profiling using desorption electrospray ionization mass spectrometry (DESI-MS) analysis. Ionization of mucosal biomass occurs directly from a standard rayon swab mounted on a rotating device and analyzed by DESI MS using an optimized protocol considering swab-inlet geometry, tip-sample angles and distances, rotation speeds, and reproducibility. Multivariate modeling of mass spectral fingerprints obtained in this way readily discriminate between different mucosal surfaces and display the ability to characterize biochemical alterations induced by pregnancy and bacterial vaginosis (BV). The method was also applied directly to bacterial biomass to confirm the ability to detect intact bacterial species from a swab. These results highlight the potential of direct swab analysis by DESI-MS for a wide range of clinical applications including rapid mucosal diagnostics for microbiology, immune responses, and biochemistry.

Mucosal Type 2 Innate Lymphoid Cells Are a Key Component of the Allergic Response to Aeroallergens.

RATIONALE: Newly characterized type 2 innate lymphoid cells (ILC2s) display potent type 2 effector functionality; however, their contribution to allergic airways inflammation and asthma is poorly understood. Mucosal biopsy used to characterize the airway mucosa is invasive, poorly tolerated, and does not allow for sequential sampling. OBJECTIVES: To assess the role of ILC2s during nasal allergen challenge in subjects with allergic rhinitis using novel noninvasive methodology. METHODS: We used a human experimental allergen challenge model, with flow cytometric analysis of nasal curettage samples, to assess the recruitment of ILC2s and granulocytes to the upper airways of subjects with atopy and healthy subjects after allergen provocation. Soluble mediators in the nasal lining fluid were measured using nasosorption. MEASUREMENTS AND MAIN RESULTS: After an allergen challenge, subjects with atopy displayed rapid induction of upper airway symptoms, an enrichment of ILC2s, eosinophils, and neutrophils, along with increased production of IL-5, prostaglandin D2, and eosinophil and T-helper type 2 cell chemokines compared with healthy subjects. The most pronounced ILC2 recruitment was observed in subjects with elevated serum IgE and airway eosinophilia. CONCLUSIONS: The rapid recruitment of ILC2s to the upper airways of allergic patients with rhinitis, and their association with key type 2 mediators, highlights their likely important role in the early allergic response to aeroallergens in the airways. The novel methodology described herein enables the analysis of rare cell populations from noninvasive serial tissue sampling.