Pulmonary Immune Cell-microbiome Interactions in the Healthy Lung

Study Overview
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Brief Summary
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The overall aim is to to provide a normal material for the composition and spatial heterogeneity of the following in the healthy lung: i) immune cell populations and their activation patterns, ii) the surrounding cytokine-chemokine milieu, including trans-compartmental fluxes of these mediators between the lung and bloodstream, and iii) the lung microbiome. Main hypotheses: Absolute and relative immune cell counts in bronchoalveolar lavage fluid (BALF) are similar to those previously reported by other methods6,7. No trans-compartmental flux of cytokines between the lungs and bloodstream is present, but cytokine concentrations (notably IL-6 and IL-8) vary with the immune-cell-microbiome composition. Immune cell (mainly T cell) activation, differentiation, and gene expression patterns are expected to differ between blood and BALF in a manner that depends on the regional diversity of the pulmonary microbiome.

Detailed Description
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The coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has brought with it several studies on the local pulmonary immune system, which was until very recently largely a terra incognita. With these new methods, apparently fundamental aspects of lung disease have been uncovered, including pulmonary hyper inflammation. However, very little is currently known about the composition of the normal immune cell population in the human lung, including its interplay with the pulmonary microbiome, that is, the commensal microbiota of interacting bacteria and fungi that reside within the healthy lung Overall design: In 50 patients (25 males, 25 females), intubated for elective surgery in general anaesthesia, an endotracheal aspirate and BAL fluid (BALF) from separate lung segments will be obtained. Furthermore, an oral and nasal swab and blood samples will be collected. This will be done immediately after intubation. The following is obtained from the patient's electronic health record: diagnosis codes and medication (type and dosage), smoking history (current/previous/never smoker; pack years), type of operation; furthermore, the health record is screened for any exclusion criteria Blood samples are drawn from the patient's peripheral venous catheter (inserted for clinical purposes) immediately before BALF collection. Bronchoscopy with BALF collection: This procedure is performed in a standardized fashion according to current clinical guidelines. Immediately prior to the procedure, an oral swab, nasal swab and an endotracheal aspirate (ETA) are obtained. FIO2 is then increased to 1.0, and the bronchoscopy procedure is performed using a disposable videoscope with an outer diameter of 5.0 mm). Three successive 50-ml aliquots of prewarmed (37°C) isotonic saline are instilled in the medial segment of the right middle lobe, aspirated immediately with low negative suction pressure (< 100 cm H2O), and pooled into a sterile glass container on ice to obtain a BALF specimen. Afterwards a mini-BAL is performed in the upper and lower lobe of the right lung with a single installation of 20 ml isotonic saline in each lobe with immediately aspiration into a sterile container. Measurements: The composition of the immune cell population, as well as the function and differentiation of various cell lines will be investigated by single-cell RNA sequencing (scRNA-seq) on selected immune cells from BALF, ETA, and blood, and this will be supplemented by bulk RNA sequencing with sample barcoding and multiplexing, giving a detailed expression pattern of all samples. The composition microbiome in BALF, ETA, and oral swabs will be assessed by targeted amplicon sequencing of the hyper-variable regions 1 through 3 of the 16S subunit of ribosomal RNA gene for bacteria. Statistical analyses: will be performed using R statistical software version 4.1.1 (R Project for Statistical Computing) within RStudio statistical software version 1.4.1717 (RStudio), and p<0.05 considered statistically significant. Inspection of normality and variance homogeneity will be done by creating qq-plots and histograms. The statistical inference tools SPIEC-EASI and HeatMaps will be used, and based on correlational analyses, principal component analyses, including non-hierarchal cluster analysis, will be applied to identify traits.

Official Title
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Pulmonary Immune Cell-microbiome Interactions in the Healthy Lung: The ILLUMINA-2 Study

Conditions
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Healthy

Intervention / Treatment
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* Procedure: Bronchoalveolar lavage


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Men and women Age 40 to 75 Exclusion Criteria: Immune deficiency Lung disease Active cancer or infection Absolute contraindications for bronchoscopy Untreated malignant arrhythmia Documented or suspected intracranial hypertension (intracranial pressure ≥ > 15 mmHg) One-lung ventilation Severe coagulopathy

Ages Eligible for Study
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Minimum Age: 40 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Women<br> | Procedure: Bronchoalveolar lavage<br>* Bronchoalveolar lavage in the middle lobe of the right lung and a mini-bronchoalveolar lavage in the upper and lower lobe of the right lung<br>|
| Men<br> | Procedure: Bronchoalveolar lavage<br>* Bronchoalveolar lavage in the middle lobe of the right lung and a mini-bronchoalveolar lavage in the upper and lower lobe of the right lung<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Lung microbiome | 16S ribosomal RNA (rRNA) and 18S rRNA PCR for bacterial or fungal pathogen identification in bronchoalveolar lavage fluid | Day 0 (subsequent to study inclusion) | 
| Lymphocyte populations | Cell populations and subpopulations evaluated by 10 colored flow cytometry (B cells, T cells, TCR subsets, Tregs/Th17, dendritic cells, myeloid cells and neutrophils) in bronchoalveolar lavage fluid and blood | Day 0 (subsequent to study inclusion) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Cell differential counts and cytomorphological analyses of BALF |  | Day 0 (subsequent to study inclusion) | 
| Trans-compartmental fluxes | (calculated from plasma- and urea-adjusted BAL) | Day 0 (subsequent to study inclusion) | 
| Auto-antibodies against tI-IFNs in blood | Measured in bronchoalveolar lavage fluid | Day 0 (subsequent to study inclusion) | 
| White blood cells counts | Total white blood cells, neutrocytes, lymphocytes, and monocytes in bronchoalveolar lavage fluid and blood | Day 0 (subsequent to study inclusion) | 
| Cytokines | Multiplex assay for measuring cytokines in bronchoalveolar lavage fluid and plasma (e.g. IL-1-beta, IL-1RA, IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-33, IL-35, TGF-beta, TNF-alpha, HMGB1) | Day 0 (subsequent to study inclusion) | 
| Number and characterizations of respiratory pathogens | Respiratory film array PCR for testing for number of pathogens | Day 0 (subsequent to study inclusion) | 
| Number and characterizations of microorganisms | Growth of pathogenic microorganisms in body fluids (e.g. urine, blood, bronchoalveolar lavage fluid) in microbiological assays | Up to 12 weeks | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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healthy lung, lung microbiota, microbiome