A method for 
INTRODUCTION Investigative bronchoscopic sampling of fluid and tissue has long been used for the in vivo evaluation of cellular activity and airway morphology (Godard et al., ; Lozewicz et al., ), and has been shown to be well‐tolerated in asthma patients (Moore et al., ). Bronchoalveolar lavage (BAL), histochemical (HC) and immunohistochemical (IHC) stains and morphological measures of biopsies obtained via bronchoscopy enable a range of specific measurements which are associated with pathological response (Kelly et al., ). However, this technique traditionally takes place without prior knowledge of the presence or absence of airway obstruction within the region undergoing sampling. Evaluating localized cellular and morphological phenomena associated with segmental airway obstruction may provide insights into the pathophysiology of chronic obstructive lung disease and aid in the development of targeted therapies. Hyperpolarized (HP) gas magnetic resonance imaging (MRI) provides a means of directly visualizing gas distribution in the lungs (Kauczor et al., ; Moller et al., ) without the use of ionizing radiation. Defect extent is commonly measured as the ventilation defect percentage (VDP) (1), which, in asthma, is spatially heterogeneous (Aysola et al., ; Zha et al., ), related to computed tomography (CT) structural abnormalities (i.e., air trapping, airway wall thickness) (Carey et al., ; Fain et al., ; Svenningsen et al., ), and associated with and predictive of exacerbations (Mummy et al., ; Mummy, Kruger, et al., ); ventilation defects have previously been shown to be spatially and quantitatively reproducible over the short (same‐day, 7‐day) (Mathew et al., ; Niles et al., ) and long‐term (7 years) (Eddy et al., ). Moreover, evidence of granulocytic inflammation is highly correlated with both global (Svenningsen et al., ) and lobar VDP (Fain et al., ). The purpose of this work was to prospectively use HP 3 He MRI to obtain paired bronchoscopy samples from ventilation defect and well‐ventilated lung regions within the same subject in a pilot population of patients who had experienced a recent viral‐induced exacerbation. Some of these findings have been previously presented in the form of two abstracts (Mummy et al., ; Mummy, Kehoe, et al., ).   MATERIALS AND METHODS 2.1 Participants and study design Participants aged 16–60 years provided written informed consent to the Viral‐Induced Asthma Exacerbation (VIAX) (Denlinger et al., ) longitudinal study. This prospective study was compliant with the Health Insurance Portability and Accountability Act (HIPAA) and was approved by the University of Wisconsin Health Sciences Institutional Review Board (IRB H‐2005‐0070). Image‐guided bronchoscopy was performed based on the presence of a defect after recovery from a virus‐induced exacerbation with the intent of sampling regions at the leading edge of airway injury. Participants underwent 1 H (standard proton) and 3 He MRI, CT, and spirometry at least 6 weeks post‐viral exacerbation and were excluded if they reported other respiratory complications (i.e., current smoker or significant smoking history (exceeding 5 pack years), pulmonary edema, atelectasis, upper airway obstruction). Bronchoscopy was performed to obtain BAL and bronchial wall biopsy samples within 30 days following imaging. 2.2 Image acquisition and analysis Anatomical 1 H and HP 3 He MRI were acquired under breath‐hold conditions by using a 1.5 Tesla Signa HDx scanner (GE Healthcare, Milwaukee WI). 3 He was polarized using a Polarean IGI 9600 hyperpolarizer (Polarean Inc., Durham NC) and mixed with N 2 such that the total volume of 3 He and N 2 was 14% of the participant's total lung capacity. The gas mixture was prepared and delivered in a Tedlar bag (Jensen Inert Products, Coral Springs FL). Participants were positioned supine with the gas dose inhaled from functional residual capacity. Ventilation defects were located anatomically using the corresponding slice from a 1 H MRI and registered CT reference (Figure ). Defects were visually identified as regions of no or low signal on an axial HP 3 He MRI. Lower but not complete absence of gas signal was still defined as a visual defect in some cases (Figure ). In a subset of participants where MRI was not available, defects were located by proxy on CT by identifying regions of hyperlucency, indicative of air trapping (Figure ). MR images were segmented and quantified using custom software tools implemented in MATLAB (MathWorks, Natick MA) as described previously (Zha et al., ). Whole lung VDP was calculated as the ventilation defect volume normalized to the total 1 H thoracic cavity volume. Chest CT was performed at end expiration (functional residual capacity) on a 64 slice multi‐detector scanner (VCT 64, GE Healthcare). Effective CT dose was adjusted to mitigate radiation exposure and maintain image quality (Sieren et al., ). Air trapping was quantified from CT images as the relative area less than −850 (RA −850 ) Hounsfield units (HU). 2.3 Bronchoscopy and analysis BAL and endobronchial biopsy samples of the airway wall were obtained via image‐guided bronchoscopy at both defect and control sites within the same or contralateral lung of a given participant. BAL samples were analyzed for differential cell counts. Oxylipin mediators contribute to the transition between pro‐inflammatory and pro‐resolution states (Tavares et al., ; Townsend et al., ) thus, oxylipin levels with and without normalization for total protein concentration in the BAL fluid were also measured. Markers of interest are shown in Table and included lipoxin A4 (LXA4) and 15‐epimer (15‐epi) of LXA4 (inhibit neutrophilic infiltration and production of pro‐inflammatory cytokines and chemokines (Bonnans et al., ; Takano et al., )) and leukotriene B4 (induces recruitment and activation of granulocytes and monocytes (Crooks & Stockley, )). Biopsy samples were embedded in paraffin and frozen before being sectioned and mounted on slides (Kelly et al., ). Biopsy samples were stained with H&E and reviewed by an expert pulmonary pathologist. A subset of participants was selected for further HC and IHC staining (markers described in Table ) based on a visual evaluation by the pathologist of the quality of tissue biopsy presentation on H&E stains. Specifically, these samples were chosen based on the quality of the sample and a preference for sections cross‐sectional to the airway. Basement membrane thickness was measured on H&E stains using ImageJ software ( https://imagej.nih.gov/ij/ ), as shown in Figure , by two imaging scientists (KJC and SK) under pathologist guidance. In each sample, approximately 20 measurements were obtained from areas both with and without epithelium, as available. The measurements were averaged to determine the mean wall thickness for that sample. Image‐based quantitative analysis of HC and IHC stained sections was performed using Aperio ImageScope software ( https://www.leicabiosystems.com/digital‐pathology/manage/aperio‐imagescope/ ). Regions of interest (ROI) of a standard size were defined on H&E stains for each sample. These ROIs were transferred to equivalent areas in HC/IHC stained slides from adjacent sections by the scientist performing image‐based measurements (KJC and SK). Cell counts were then obtained and normalized to total ROI area. 2.4 Statistical analysis Normality was evaluated using Shapiro–Wilk tests, which showed that data was not normally distributed. Differences in BAL fluid cell counts, oxylipin levels as well as basement membrane thickness in defect versus control sites were evaluated using Wilcoxon Signed Rank tests. Univariate relationships were evaluated using Spearman correlations. Observable differences in cell counts for each participant were defined as a factor of two increase or decrease between defect and control sites. Data are presented as the mean ± standard deviation unless otherwise specified. All statistical analysis was performed using SPSS (Version 29.0, IBM). p values less than 0.05 were considered statistically significant.   Participants and study design Participants aged 16–60 years provided written informed consent to the Viral‐Induced Asthma Exacerbation (VIAX) (Denlinger et al., ) longitudinal study. This prospective study was compliant with the Health Insurance Portability and Accountability Act (HIPAA) and was approved by the University of Wisconsin Health Sciences Institutional Review Board (IRB H‐2005‐0070). Image‐guided bronchoscopy was performed based on the presence of a defect after recovery from a virus‐induced exacerbation with the intent of sampling regions at the leading edge of airway injury. Participants underwent 1 H (standard proton) and 3 He MRI, CT, and spirometry at least 6 weeks post‐viral exacerbation and were excluded if they reported other respiratory complications (i.e., current smoker or significant smoking history (exceeding 5 pack years), pulmonary edema, atelectasis, upper airway obstruction). Bronchoscopy was performed to obtain BAL and bronchial wall biopsy samples within 30 days following imaging.   Image acquisition and analysis Anatomical 1 H and HP 3 He MRI were acquired under breath‐hold conditions by using a 1.5 Tesla Signa HDx scanner (GE Healthcare, Milwaukee WI). 3 He was polarized using a Polarean IGI 9600 hyperpolarizer (Polarean Inc., Durham NC) and mixed with N 2 such that the total volume of 3 He and N 2 was 14% of the participant's total lung capacity. The gas mixture was prepared and delivered in a Tedlar bag (Jensen Inert Products, Coral Springs FL). Participants were positioned supine with the gas dose inhaled from functional residual capacity. Ventilation defects were located anatomically using the corresponding slice from a 1 H MRI and registered CT reference (Figure ). Defects were visually identified as regions of no or low signal on an axial HP 3 He MRI. Lower but not complete absence of gas signal was still defined as a visual defect in some cases (Figure ). In a subset of participants where MRI was not available, defects were located by proxy on CT by identifying regions of hyperlucency, indicative of air trapping (Figure ). MR images were segmented and quantified using custom software tools implemented in MATLAB (MathWorks, Natick MA) as described previously (Zha et al., ). Whole lung VDP was calculated as the ventilation defect volume normalized to the total 1 H thoracic cavity volume. Chest CT was performed at end expiration (functional residual capacity) on a 64 slice multi‐detector scanner (VCT 64, GE Healthcare). Effective CT dose was adjusted to mitigate radiation exposure and maintain image quality (Sieren et al., ). Air trapping was quantified from CT images as the relative area less than −850 (RA −850 ) Hounsfield units (HU).   Bronchoscopy and analysis BAL and endobronchial biopsy samples of the airway wall were obtained via image‐guided bronchoscopy at both defect and control sites within the same or contralateral lung of a given participant. BAL samples were analyzed for differential cell counts. Oxylipin mediators contribute to the transition between pro‐inflammatory and pro‐resolution states (Tavares et al., ; Townsend et al., ) thus, oxylipin levels with and without normalization for total protein concentration in the BAL fluid were also measured. Markers of interest are shown in Table and included lipoxin A4 (LXA4) and 15‐epimer (15‐epi) of LXA4 (inhibit neutrophilic infiltration and production of pro‐inflammatory cytokines and chemokines (Bonnans et al., ; Takano et al., )) and leukotriene B4 (induces recruitment and activation of granulocytes and monocytes (Crooks & Stockley, )). Biopsy samples were embedded in paraffin and frozen before being sectioned and mounted on slides (Kelly et al., ). Biopsy samples were stained with H&E and reviewed by an expert pulmonary pathologist. A subset of participants was selected for further HC and IHC staining (markers described in Table ) based on a visual evaluation by the pathologist of the quality of tissue biopsy presentation on H&E stains. Specifically, these samples were chosen based on the quality of the sample and a preference for sections cross‐sectional to the airway. Basement membrane thickness was measured on H&E stains using ImageJ software ( https://imagej.nih.gov/ij/ ), as shown in Figure , by two imaging scientists (KJC and SK) under pathologist guidance. In each sample, approximately 20 measurements were obtained from areas both with and without epithelium, as available. The measurements were averaged to determine the mean wall thickness for that sample. Image‐based quantitative analysis of HC and IHC stained sections was performed using Aperio ImageScope software ( https://www.leicabiosystems.com/digital‐pathology/manage/aperio‐imagescope/ ). Regions of interest (ROI) of a standard size were defined on H&E stains for each sample. These ROIs were transferred to equivalent areas in HC/IHC stained slides from adjacent sections by the scientist performing image‐based measurements (KJC and SK). Cell counts were then obtained and normalized to total ROI area.   Statistical analysis Normality was evaluated using Shapiro–Wilk tests, which showed that data was not normally distributed. Differences in BAL fluid cell counts, oxylipin levels as well as basement membrane thickness in defect versus control sites were evaluated using Wilcoxon Signed Rank tests. Univariate relationships were evaluated using Spearman correlations. Observable differences in cell counts for each participant were defined as a factor of two increase or decrease between defect and control sites. Data are presented as the mean ± standard deviation unless otherwise specified. All statistical analysis was performed using SPSS (Version 29.0, IBM). p values less than 0.05 were considered statistically significant.   RESULTS The Consolidated Standards of Reporting Trials diagram in Figure shows that of the 66 participants enrolled in the cold virus‐induced exacerbation study as detailed previously (Denlinger et al., ), 11 (5 females, 6 males; 9 asthma; age, 22.8 ± 3.4 years) underwent image‐guided bronchoscopy. Participants in this study were recruited based on having a recent exacerbation due to a detected rhinovirus ( N = 7) or respiratory syncytial virus ( N = 2) in the upper airways 6–8 week prior to HP gas MRI. Table provides a summary of participant demographics, pulmonary function, and imaging measurements acquired just prior to image‐guided bronchoscopy. Briefly, the mean forced expiratory volume in the first second (FEV 1 ) and FEV 1 to forced vital capacity ratio (FEV 1 /FVC) were in the normal range, while whole lung VDP (Pike et al., ) and RA −850 (Mets et al., ) measures were typical of those seen in mild asthma or healthy normal participants. Bronchoscopy was performed 11 ± 8 days after imaging (median [min–max] = 9 [2–29] days), which was also 73 ± 21 days since the onset of cold symptoms (median [min–max] = 71 [51–129] days). All 11 participants had BAL performed in both the defect and control segments and 8 had endobronchial biopsies collected. Biopsy samples from four of these participants met quality standards and also underwent HC and IHC analysis. 3.1 BAL results Figure shows differences in cell counts and oxylipin mediators from BAL fluid. Neutrophil (defect: 0.27 ± 0.18 × 10 6 cells, control: 0.17 ± 0.19 × 10 6 cells; p = 0.06) and total granulocyte counts (defect: 0.32 ± 0.22 × 10 6 cells, control: 0.19 ± 0.19 × 10 6 cells; p = 0.08) had a trend for a difference in defect as compared to control sites; eosinophil cell counts were not different between sites ( p = 0.1). Figure further shows that eicosanoid lipoxin A4 (LXA4) was significantly less (defect: 69 ± 14 μg/mL, control: 76 ± 14 μg/mL; p = 0.05) while both 15‐epimer (15‐epi) of LXA4 (defect: 141 ± 52 μg/mL, control: 111 ± 61 μg/mL; p = 0.04) and 15‐epi‐LXA4 normalized to the total protein concentration (defect: 1.4 ± 0.5, control: 0.9 ± 0.3; p = 0.02) were significantly greater in defect compared to control sites. No differences were observed for leukotriene B4 (chemotactic for neutrophils) or cysteinyl leukotrienes (pro‐inflammatory oxylipins made by eosinophils) nor were there any differences noted for several pro‐inflammatory cytokines in the BAL (inclusive of Eotaxin, IFNg, IL‐13, IL‐5, and MCP‐1; data not shown). Relationships between pro‐resolving oxylipins and granulocyte counts are presented in Figure . We observed inverse correlations between average BAL eosinophil concentration with normalized LXA4 ( ρ = −0.70, p = 0.04), and between the average normalized 15‐epi‐LXA4 with neutrophils ( ρ = −0.80, p = 0.01) and macrophages ( ρ = −0.77, p = 0.02); average eosinophils and normalized leukotriene B4 trended towards an association ( ρ = −0.59, p = 0.09). 3.2 Biopsy findings Figure shows CD4, Periodic Acid‐Schiff (PAS), and Masson's Trichrome stains of paired defect‐control biopsy samples in two participants. The CD4 stain shows a substantial increase in CD4 cells (stained brown) in defect versus control site. PAS shows increased mucin and goblet cell hyperplasia (blue and purple) in the defect site. Finally, Trichrome stain shows qualitative evidence of squamous metaplasia, loss of epithelial cilia, and collagen deposition (blue) characteristic of chronic airway injury and fibrosis. Basement membrane wall thickness was similar for tissue biopsies from defect versus control sites (6.97 ± 1.71 microns vs. 6.43 ± 2.54 microns, respectively; p = 0.9). Both participant 42 and 126 had evidence of elevated CD8, with participant 126 also demonstrating elevated CD4, CD10, and CD68, in defect as compared to control sites. There were no observable differences for participant 96 and 110 between defect and control sites for the cell marker stains evaluated.   BAL results Figure shows differences in cell counts and oxylipin mediators from BAL fluid. Neutrophil (defect: 0.27 ± 0.18 × 10 6 cells, control: 0.17 ± 0.19 × 10 6 cells; p = 0.06) and total granulocyte counts (defect: 0.32 ± 0.22 × 10 6 cells, control: 0.19 ± 0.19 × 10 6 cells; p = 0.08) had a trend for a difference in defect as compared to control sites; eosinophil cell counts were not different between sites ( p = 0.1). Figure further shows that eicosanoid lipoxin A4 (LXA4) was significantly less (defect: 69 ± 14 μg/mL, control: 76 ± 14 μg/mL; p = 0.05) while both 15‐epimer (15‐epi) of LXA4 (defect: 141 ± 52 μg/mL, control: 111 ± 61 μg/mL; p = 0.04) and 15‐epi‐LXA4 normalized to the total protein concentration (defect: 1.4 ± 0.5, control: 0.9 ± 0.3; p = 0.02) were significantly greater in defect compared to control sites. No differences were observed for leukotriene B4 (chemotactic for neutrophils) or cysteinyl leukotrienes (pro‐inflammatory oxylipins made by eosinophils) nor were there any differences noted for several pro‐inflammatory cytokines in the BAL (inclusive of Eotaxin, IFNg, IL‐13, IL‐5, and MCP‐1; data not shown). Relationships between pro‐resolving oxylipins and granulocyte counts are presented in Figure . We observed inverse correlations between average BAL eosinophil concentration with normalized LXA4 ( ρ = −0.70, p = 0.04), and between the average normalized 15‐epi‐LXA4 with neutrophils ( ρ = −0.80, p = 0.01) and macrophages ( ρ = −0.77, p = 0.02); average eosinophils and normalized leukotriene B4 trended towards an association ( ρ = −0.59, p = 0.09).   Biopsy findings Figure shows CD4, Periodic Acid‐Schiff (PAS), and Masson's Trichrome stains of paired defect‐control biopsy samples in two participants. The CD4 stain shows a substantial increase in CD4 cells (stained brown) in defect versus control site. PAS shows increased mucin and goblet cell hyperplasia (blue and purple) in the defect site. Finally, Trichrome stain shows qualitative evidence of squamous metaplasia, loss of epithelial cilia, and collagen deposition (blue) characteristic of chronic airway injury and fibrosis. Basement membrane wall thickness was similar for tissue biopsies from defect versus control sites (6.97 ± 1.71 microns vs. 6.43 ± 2.54 microns, respectively; p = 0.9). Both participant 42 and 126 had evidence of elevated CD8, with participant 126 also demonstrating elevated CD4, CD10, and CD68, in defect as compared to control sites. There were no observable differences for participant 96 and 110 between defect and control sites for the cell marker stains evaluated.   DISCUSSION We performed a novel image‐guided bronchoscopy approach using HP 3 He MRI to sample sites of ventilation defect paired with well‐ventilated control sites within the lungs of the same patient in a pilot cohort of patients who had recently experienced a viral‐induced exacerbation. Prospectively identified areas of ventilation defects which are associated with airway inflammation and obstruction exhibited qualitative evidence of goblet cell hyperplasia and squamous metaplasia; trends suggesting greater numbers of neutrophils and granulocytes in defect regions; and significant differences in immune cell modifying proteins in defect regions compared to well‐ventilated control regions. In some instances, p ‐values suggest mean differences that do not meet the traditional p < 0.05 threshold. These trends are maintained and reported to guide future work as our study is almost certainly underpowered due to the challenges of recruiting patients to imaging and bronchoscopy within a window that is shortly after viral‐induced asthma exacerbation. Future studies may benefit from further study of these trends. Lipoxin protein levels including LXA4 and 15‐epi‐LXA4 were significantly different in defect versus control sites. Oxylipin mediators are derived from plasma cell membranes and contribute to the transition between pro‐inflammatory and pro‐resolution states (Tavares et al., ; Townsend et al., ). Lipoxins play an important role in the downregulation of inflammation, with 15‐epi‐LXA4 having been previously shown to facilitate the resolution of acute lung inflammation (Sekheri et al., ). In addition, the number of neutrophils and granulocytes trended towards an increase in defect regions. The presence of granulocytes in asthmatic airways is linked to increased disease severity and airway inflammation, and exacerbations (Fahy, ). Together, these findings are consistent with the immunomodulatory role of these proteins and strengthen earlier retrospective results linking ventilation defects with local inflammatory response (Fain et al., ). In a subset of 4 participants, we conducted extensive analysis of a suite of HC and IHC markers of inflammation, injury, and cellular proliferation. In one such participant, PAS staining exhibited a visually striking increase in mucin and goblet cell hyperplasia in the defect region that included quantitative increases in CD4, CD8, CD10, and CD68 staining in the same biopsy sample. The presence of CD4 or CD8 cells in bronchial wall biopsies of asthma patients has been linked to a longitudinal loss of lung function measured via spirometry (den Otter et al., ). Another participant demonstrated an obviously stratified epithelial layer with squamous metaplasia and a marked increase in collagen in the tissue deep to the basement membrane in the tissue biopsy from the defect site, alterations which are characteristic of chronic airway injury and repair. Interestingly, this participant showed no observable differences in quantitative stains for cellular activity from the same site, which suggests that the observed pathophysiology in this participant may be the result of past chronic airway injury and inflammation. The difference in inflammatory cell counts and mediators between defect and control sites as well as evidence of abnormal pathology within defect regions demonstrate the utility of functional image‐guided bronchoscopy, suggesting the potential for image‐guided treatment interventions. In fact, asthma patients treated using image‐guided bronchial thermoplasty using HP gas MRI (Hall et al., ; Svenningsen et al., ) have similar improvements in quality of life as compared to patients treated using standard bronchial thermoplasty protocols, but significantly reduced number of radiofrequency activations. Future clinical practice which combines the ability to identify and localize abnormally functioning airway segments and evaluate the underlying pathology to treat with phenotype‐specific regimens and image‐guided interventions could result in improved outcomes in asthma patients. Important limitations to this work include the small sample size, especially in the N = 4 subset of participants in which HC/IHC staining was performed. Histology sections were further limited in sample size in this study given the airway epithelium integrity was not intact in most of the biopsy samples acquired. Moreover, such qualitative differences are also prone to sampling bias within a given tissue section. Although the generalizability of these findings is limited, there is still value in comparing paired defect and control sites for sections where control and defect epithelium are both intact as shown in Figure . More robust methods of biopsy tissue sampling and handling are currently being used for ongoing studies in severe asthma. In addition, HP 3 He MRI was not acquired in three participants and thus, regions of CT air trapping were chosen as defect sites. Previous work, however, has demonstrated a spatial relationship between MRI ventilation defects and CT air trapping (Carey et al., ; Fain et al., ), hence, regions of air trapping identified using CT are likely a good estimate and surrogate for 3 He MRI ventilation defect regions. Hyperpolarized MRI studies in the lungs have also largely replaced 3 He gas with 129 Xe gas, which has slightly different volumes of distribution that could have small but measurable effects on airway defect size (Stewart et al., ). Finally, as MRI and CT air trapping were acquired only following an exacerbation, it is difficult to discern whether the ventilation defects selected for image‐guided bronchoscopy in these patients were related to active airway inflammation in response to the viral‐induced exacerbation or whether they were the consequence of prior airway injury. We would also like to note that this study was performed before CT‐image guided bronchoscopy was implemented into routine clinical practice. 4.1 Conclusion In this study, we have demonstrated the feasibility and utility of bronchoscopy guided by functional imaging. In 11 participants who had recently experienced a virus‐induced exacerbation, lung regions with abnormal airway function identified via HP gas MRI exhibited an increased presence of inflammatory cells and mediators as compared to well‐functioning lung regions. Results in this pilot population suggest that ventilation patterns observed via HP gas MRI provide a valuable tool for the identification and evaluation of localized airway phenomena associated with regional reductions in lung function. Advancing the understanding of airway activity specifically associated with localized regions of airway obstruction in future investigations may help refine the definition of asthma phenotypes and guide the development and evaluation of personalized, targeted therapies.   Conclusion In this study, we have demonstrated the feasibility and utility of bronchoscopy guided by functional imaging. In 11 participants who had recently experienced a virus‐induced exacerbation, lung regions with abnormal airway function identified via HP gas MRI exhibited an increased presence of inflammatory cells and mediators as compared to well‐functioning lung regions. Results in this pilot population suggest that ventilation patterns observed via HP gas MRI provide a valuable tool for the identification and evaluation of localized airway phenomena associated with regional reductions in lung function. Advancing the understanding of airway activity specifically associated with localized regions of airway obstruction in future investigations may help refine the definition of asthma phenotypes and guide the development and evaluation of personalized, targeted therapies.   DGM was responsible for developing the image guidance workflow, image processing, analysis, and interpretation and for preparing the first draft of the manuscript. KJC was responsible for data collection, image analysis and pathology interpretation. MJM was responsible for statistical analysis, interpretation of the results and writing and editing the manuscript. SK was responsible for data collection and biopsy sample analysis. SE was responsible for developing methodology and recommended cell‐marker stains for tissue pathology, and for processing and analysis of BAL lipid protein analysis. MWJ was responsible for developing methodology and recommended cell‐marker stains for tissue pathology, and interpretation of the results. MDE was responsible for statistical analysis and design. RLS was responsible for developing the study design and interpretation of the results. MS was responsible for interpretation of the results. NNJ was responsible for developing the study design, supervising the bronchoscopy protocol and interpretation of the results. LCD was responsible for developing the study design, performing bronchoscopy procedures and interpretation of the results. SBF was responsible for developing the study design, interpretation of the results, and direction of all aspects of the study, and writing and editing the manuscript. All authors had an opportunity to review and revise the manuscript and approved its final submitted version.   This study was funded by National Heart, Lung, and Blood Institute (NHLBI) grant R01 HL080412 (Jarjour) with institutional resources funded by National Center for Advancing Translational Sciences (NCATS) grant 1 UL1RR025011. Secondary analyses were supported by R01 HL115118 (Denlinger) with effort contributed by The William W. and Judith H. Busse Professorship in Allergy and Asthma Research. SBF is supported by the National Institutes of Health (NIH) R01 HL126771, R01 HL146689, and R01 HL169765.   DGM and SBF serve as scientific advisors for Polarean LLC. SBF also receives research funding from Polarean LLC, Siemens Healthineers INC, and GE Healthcare INC for development of pulmonary CT and MRI methods. MWJ has received research funding from Hoffmann‐La Roche (unrelated to this work). LCD reports consultation with GlaxoSmithKline, AstraZeneca, Sanofi, and OM Pharma during the course of this study. MJM, KJC, SK, SE, MDE, RLS, MS and NNJ have no disclosures.   ETHICS STATEMENT This human study was approved by University of Wisconsin Health Sciences Institutional Review Board ‐ approval: (IRB H‐2005‐0070). All participants provided written informed consent to participate in this study.   