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American Journal of Respiratory and Critical Care Medicine logoLink to American Journal of Respiratory and Critical Care Medicine
. 2022 May 13;206(3):260–270. doi: 10.1164/rccm.202109-2133OC

Characterization of Immunopathology and Small Airway Remodeling in Constrictive Bronchiolitis

Sergey S Gutor 1,✉, Bradley W Richmond 1,2, Rui-Hong Du 1, Pingsheng Wu 1,3, Jae Woo Lee 4, Lorraine B Ware 1,5, Ciara M Shaver 1, Sergey V Novitskiy 1, Joyce E Johnson 5, John H Newman 1, Stephen I Rennard 6, Robert F Miller 1, Timothy S Blackwell 1,2, Vasiliy V Polosukhin 1
PMCID: PMC9890264  PMID: 35550018

Abstract

Rationale

Constrictive bronchiolitis (ConB) is a relatively rare and understudied form of lung disease whose underlying immunopathology remains incompletely defined.

Objectives

Our objectives were to quantify specific pathological features that differentiate ConB from other diseases that affect the small airways and to investigate the underlying immune and inflammatory phenotype present in ConB.

Methods

We performed a comparative histomorphometric analysis of small airways in lung biopsy samples collected from 50 soldiers with postdeployment ConB, 8 patients with sporadic ConB, 55 patients with chronic obstructive pulmonary disease, and 25 nondiseased control subjects. We measured immune and inflammatory gene expression in lung tissue using the NanoString nCounter Immunology Panel from six control subjects, six soldiers with ConB, and six patients with sporadic ConB.

Measurements and Main Results

Compared with control subjects, we found shared pathological changes in small airways from soldiers with postdeployment ConB and patients with sporadic ConB, including increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and lymphocyte infiltration. Using principal-component analysis, we showed that ConB pathology was clearly separable both from control lungs and from small airway disease associated with chronic obstructive pulmonary disease. NanoString gene expression analysis from lung tissue revealed T-cell activation in both groups of patients with ConB with upregulation of proinflammatory pathways, including cytokine–cytokine receptor interactions, NF-κB (nuclear factor-κB) signaling, TLR (Toll-like receptor) signaling, T-cell receptor signaling, and antigen processing and presentation.

Conclusions

These findings indicate shared immunopathology among different forms of ConB and suggest that an ongoing T-helper cell type 1–type adaptive immune response underlies airway wall remodeling in ConB.

Keywords: chronic obstructive pulmonary disease, small airway disease, inflammation, gene expression profiling

At a Glance Commentary

Scientific Knowledge on the Subject

Constrictive bronchiolitis (ConB) is a relatively understudied form of small airway disease characterized by narrowing or obliteration of bronchioles due to subepithelial fibrosis. Although ConB has been associated with a variety of environmental or occupational exposures, autoimmune diseases, and transplantation of solid organs or hematopoietic stem cells, the spectrum of pathology in ConB is incompletely characterized, and the underlying immune and inflammatory phenotype remains poorly understood.

What This Study Adds to the Field

Our study shows a conserved immunopathological phenotype in sporadic and deployment-related ConB, which is separable from small airway disease associated with chronic obstructive pulmonary disease. In addition to collagen deposition in small airway walls, ConB is characterized by smooth muscle hypertrophy and an influx of T and B lymphocytes. Findings of T-cell activation in the lungs of patients with ConB suggest that chronic adaptive immune activation contributes to small airway pathology in this disease.

“Bronchiolitis” is a term used to define the presence of acute or chronic inflammation in small distal airways that are <2 mm in internal diameter and have no cartilage or submucosal glands (1–4). This spectrum of diseases includes acute bronchiolitis, constrictive bronchiolitis (ConB), bronchiolitis obliterans and organizing pneumonia, respiratory bronchiolitis, follicular bronchiolitis, and diffuse panbronchiolitis (2). ConB, or obliterative bronchitis, is a relatively understudied form of small airway disease characterized by subepithelial fibrosis, extrinsic compression, and sometimes obliteration of bronchioles (5–8). ConB can be caused by occupational exposure to toxic and irritating substances, including fumes, vapors, or gases (9, 10). In addition, ConB can occur in association with autoimmune diseases (11, 12), or after solid organ transplantation (particularly lung) or hematopoietic stem cell transplantation (13–18). Sporadic ConB can also occur without a defined exposure (19).

The earliest cases of ConB were reported after exposure to sulfur dioxide (SO2), hydrogen sulfide, and sulfur mustard gases used during World War I (7). Further studies suggested that luminal obstruction by either fibroproliferative polyps (e.g., after nitrogen dioxide exposure) or fibrotic narrowing (e.g., after SO2 exposure) could occur as manifestations of small airway disease from exposures during warfare (5, 20). More recently, biopsy-confirmed case series of ConB have been described in Iranian survivors of sulfur mustard gas attacks (21) and in active-duty U.S. military personnel after deployment to Southwest Asia (22–30). These U.S. soldiers reported deployment-associated respiratory symptoms such as persistent cough, exertional dyspnea, and exercise intolerance (22–37). In this group of symptomatic soldiers deployed to Southwest Asia, we recently reported a detailed histological analysis of lung biopsy samples confirming the presence of ConB and identifying diffuse inflammatory and fibrotic changes in the distal lungs that involved small airways, alveolar parenchyma, and pleura (38). These pathological changes were accompanied by hypertensive-type vascular remodeling in bronchovascular bundles surrounding small airways (38).

In this study, we sought to quantify specific immunopathological features that differentiate ConB from other diseases that affect the small airways. To accomplish this goal, we performed a comprehensive morphometric analysis of small airways in lung biopsy samples collected from soldiers with ConB, patients with sporadic ConB, patients with chronic obstructive pulmonary disease (COPD), and control subjects without chronic lung disease. After quantifying pathological features, we investigated the underlying immune and inflammatory phenotype using gene expression profiling. These studies identified a conserved pathological and immune and inflammatory phenotype in ConB and implicated shared biologic mechanisms between sporadic and deployment-related ConB. Some of the results of these studies have been previously reported in the form of abstracts (39, 40).

Methods

Study Design

We evaluated biopsy samples from 50 U.S. soldiers with histories of deployment in Southwest Asia and/or Afghanistan who received diagnoses of ConB on surgical lung biopsies. In these patients, diagnoses of ConB were entered into their medical records on the basis of evaluation by a clinical pathologist who identified the presence of visible subepithelial fibrosis that surrounds, rather than fills, the bronchiolar lumen (2, 41). In addition, lung sections from eight patients with diagnoses of sporadic ConB were obtained from explanted lungs at the time of lung transplantation. We also obtained lung specimens from 55 former smokers with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I–IV COPD and 25 lifelong nonsmokers without histories of chronic lung disease (nondiseased [ND] control subjects). Seventeen tissue specimens in the ND group came from deceased organ donors whose lungs were rejected for transplantation but were procured for research at the University of California, San Francisco, Medical Center. Lung tissue specimens from 8 ND control subjects and 31 patients with GOLD stages I–III COPD were obtained at the time of lung cancer resection. Airways from tissue outside the tumor margins were analyzed for these subjects. Explanted lungs from 24 patients with GOLD stage IV COPD were obtained at the time of lung transplantation. All samples (except for the 17 samples from the University of California, San Francisco) were obtained from the pathology archive at Vanderbilt University Medical Center (collected from 2004 to 2018).

Table 1 summarizes the demographic and clinical characteristics of the study participants. Lung specimens were collected, and this study was performed with approval from the Institutional Review Board at Vanderbilt University Medical Center (protocol numbers 060162, 000616, and 161046).

Table 1.

Clinical Characteristics of Study Participants

  ND Control Subjects (n = 25) Soldiers with ConB (n = 50) Patients with Sporadic ConB (n = 8) Patients with COPD by GOLD Stage
Stages I and II (n = 21) Stages III and IV (n = 34)
FEV1, % of predicted 103* (88–109) 87 (64–123) 29 (17–86) 72 (53–95) 24 (12–48)
FEV1/FVC, % of FVC 79* (74–84) 79 (64–117) 78 (26–87) 58 (40–68) 33 (21–53)
DlCO 92.5* (61–117) 86.5 (61–125) 38.5 (33–95) 78 (46–111) 30 (7–73)
Age, yr 46 (21–69) 36 (26–55) 59 (45–66) 66 (58–77) 57.5 (43–71)
Sex          
 Male 17 (68) 47 (94) 4 (50) 11 (52) 15 (44)
 Female 8 (32) 3 (6) 4 (50) 10 (48) 19 (56)
Smoking history          
 Lifelong nonsmokers 25 (100) 33 (66) 3 (37) — —
 Current smokers — 3 (6) — — —
 Former smokers — 14 (28) 5 (63) 21 (100) 34 (100)
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Definition of abbreviations: ConB = constrictive bronchiolitis; COPD = chronic obstructive pulmonary disease; GOLD = Global Initiative for Chronic Obstructive Lung Disease; ND = nondiseased.

Data are expressed as median (range) or n (%).

*

Partially available data (n = 8).

Histopathological Analysis and Morphometry

All tissue samples were fixed with 10% buffered formalin and paraffin embedded (two tissue blocks for each study participant). Ten serial sections (5 μm) from each tissue block were made, four of which were used for hematoxylin and eosin, periodic acid–Schiff, picrosirius red, or Masson trichrome staining. Serial sections were used for immunostaining with primary antibodies against CD4 (cluster of differentiation 4) (rabbit polyclonal, ab133616 [EPR6855]; Abcam), CD8 (mouse monoclonal, GTX75393 [4B11]; GeneTex), CD19 (mouse monoclonal, GTX42325 [LE-CD19]; GeneTex), neutrophil elastase (mouse monoclonal, GTX72042 [NP57]; GeneTex), and elastin (ab237989 [ELN/1981]; Abcam) to detect T helper, T cytotoxic, and B cells, neutrophils, and elastin content, respectively.

All airways with continuous walls and <2 mm in internal diameter were examined. The average number and diameter of small airways examined per section are presented in Table E1 in the online supplement. Airways containing mural cartilage or submucosal glands were considered large airways and were excluded from the analysis. Lungs were not inflated or perfused before fixation, and all histomorphometry analyses were done according to American Thoracic Society and European Respiratory Society standards for quantitative assessment of lung structure in noninflated lung biopsy samples (42).

To assess airway wall remodeling, the area of the epithelium, lamina propria, smooth muscle, and adventitia was measured for each airway on digital microscopic images (10× objective) on sections stained with periodic acid–Schiff and then normalized to the reticular basement membrane (RBM) length. These measurements are presented as ratios of volume to surface area: epithelial height, lamina propria thickness, smooth muscle layer thickness, and adventitia thickness, respectively. Alveolar attachments were identified and counted for each small airway and normalized to RBM length. The length of the basement membrane contiguous with the adjacent pulmonary vessel and attachments to the vascular adventitia, were excluded from the measurements. The luminal area occupied by mucus plugging was calculated as the ratio of mucus content area to maximal luminal area (the area of a circle formed by the RBM length minus the area occupied by the epithelium). Collagen and elastin contents were measured as the area containing a specific fluorescence or immunofluorescence signal within the total area of the airway subepithelium on sections stained with picrosirius red or immunostained with antielastin antibodies using a red fluorescence microscope technique. Neutrophils, CD4+ cells, and CD8+ cells were enumerated within the airway walls on immunostained sections, with further normalization to the length of RBM. The presence of lymphoid follicles adjacent to small airways was assessed on sections immunostained for CD19-expressing B cells.

Morphometric measurements were made by two independent pathologists blinded to study group using Image-Pro Plus 7.0 software (Media Cybernetics) or ImageJ 1.8.0 (NIH).

RNA Isolation and Expression Analysis

RNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissue blocks from six ND control subjects, six soldiers with ConB, six patients with sporadic ConB, and six patients with COPD (GOLD stage II) who were randomly selected for these studies and had morphometry characteristics representative of the patient group (see Table E2). For selected lung biopsy samples, additional serial sections (10 μm) were cut from each FFPE tissue block and processed using truXTRAC FFPE microTUBE RNA Column Purification Kit (520161; Covaris Inc.) using the LE220-plus Focused-ultrasonicator (Covaris Inc.).

For RNA expression analysis, we used the nCounter Immunology Panel (NanoString Technologies). We performed multiplex gene expression analysis for 579 immune and inflammatory genes and 15 internal reference genes. Data analysis was done using nSolver 4.0 Analysis Software (NanoString Technologies).

Statistical Analysis

The normality of continuous variable distribution was examined using the Shapiro-Wilk test. We conducted pairwise comparisons among study groups using Student’s t test or the Mann-Whitney U test, and P values were Bonferroni adjusted for multiple comparisons. Cell type scores were calculated on the basis of RNA expression degrees of immune cell marker genes as the geometric mean of the log2-transformed normalized counts for each set of marker genes. For statistical comparison of inflammatory cell type scores, P values were calculated using the method described by Danaher and colleagues and implemented in nSolver 4.0 Analysis Software (43). For gene expression analysis, P values were Benjamini-Hochberg adjusted. Adjusted P values < 0.05 were considered to indicate statistical significance. To better visualize the variation present in our data, to identify sample similarity, and to show major contributors to variability, we performed principal-component analysis (PCA) using all measured histomorphometry parameters. All analyses were performed using R version 3.5.2 (www.r-project.org).

Results

ConB Is Characterized by Collagen Deposition and Increased Smooth Muscle Thickness in Small Airway Walls

To define specific pathological features of ConB, we compared lung specimens from soldiers with ConB, patients with severe end-stage sporadic ConB requiring lung transplantation, patients with mild to moderate COPD (GOLD stages I and II), patients with severe to very severe COPD (GOLD stages III and IV), and ND control subjects (Table 1 and Figure 1A). To quantify pathological changes in and around small airways, we measured 1) epithelial height and thickness of the lamina propria, smooth muscle, and adventitia (Figures 1B–1E), 2) collagen and elastin content of the airway wall (Figures 1F and 1G), 3) alveolar attachments (see Figure E1B), and 4) airway luminal area occupied with mucus plugs (see Figure E1A).

Figure 1.

Figure 1.

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Histomorphometric characteristics of small airways in constrictive bronchiolitis (ConB) and chronic obstructive pulmonary disease (COPD). (A) Representative small airways from a nondiseased (ND) control subject, a soldier with ConB, a patient with sporadic ConB, a patient with GOLD stages I–II COPD, and a patient with GOLD stages III–IV COPD. Top row depicts periodic acid–Schiff staining, and bottom row depicts red fluorescence after picrosirius red staining. Scale bars, 100 μm. (B–G) Box-and-whisker plots showing morphometric characteristics of small airway walls. Boxes represent the interquartile range, whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the box, circles beyond the whiskers represent extreme values, and the line within each box represents the median. Groups were compared pairwise using the Mann-Whitney U test. P values were Bonferroni adjusted for multiple comparisons. The threshold for significance was 0.05. Asterisks denote significant differences compared with ND control subjects (*P < 0.05, **P < 0.01, and ***P < 0.001), daggers denote significant differences compared with soldiers with ConB (††P < 0.01 and †††P < 0.001), and double daggers denote significant differences compared with sporadic ConB groups (‡P < 0.05, ‡‡P < 0.01, and ‡‡‡P < 0.001). GOLD = Global Initiative for Chronic Obstructive Lung Disease; VS(adv,rbm) = adventitia thickness; VS(epi,rbm) = epithelial height; VS(lp,rbm) = lamina propria thickness; VS(sm,rbm) = smooth muscle layer thickness.

Consistent findings in both groups of patients with ConB were increased smooth muscle layer thickness and increased collagen content in the airway wall compared with other groups (Figures 1D and 1F). As illustrated in Figures 1A and E2 (lower panels), thin collagen fibers within the small airway wall in ND control subjects were replaced with thick, wavy, and irregularly arranged collagen bundles in both ConB groups. In the smooth muscle layer, increased thickness appeared to be due to both smooth muscle cell hypertrophy and hyperplasia. Compared with soldiers with ConB, patients with sporadic ConB had greater lamina propria thickness and adventitial thickness (Figures 1C and 1E). No changes in epithelial height, elastin content, mucus plugging, or alveolar attachments were observed in small airways from patients with ConB compared with ND control subjects (see Table E3).

In contrast to ConB airways, small airways in patients with COPD showed more complex pathology involving each airway compartment (Figure 1; see Figure E2). Compared with ND control subjects, patients with COPD had increased epithelial height, lamina propria thickness, and adventitial thickness (Figures 1B, 1C, and 1E), together with increased mucus plugging and reduced airway attachments (see Figure E1). Compared with soldiers with ConB, patients with COPD had greater epithelial height, lamina propria thickness, and adventitia thickness, as well as reduced airway attachments (Figures 1B, 1C, 1E, and 1G; see Figure E1B). In comparing the most severe disease groups (patients with GOLD stages III and IV COPD and those with sporadic ConB), patients with GOLD stages III and IV COPD had increased epithelial height (related to goblet cell hyperplasia), reduced smooth muscle thickness and collagen content, and reduced numbers of alveolar attachments (Figures 1B, 1D, and 1F; see Figure E1B and Table E3).

In summary, small airway pathology in ConB is distinct from that in COPD and localized predominantly in the mural compartment of airway walls, consisting of collagen deposition and thickening of the smooth muscle layer.

Lymphocyte Infiltration Is Present in ConB

To compare the immune and inflammatory phenotype in small airways from patients with ConB and those with COPD, we quantified lymphocytes and neutrophils and the percentage of small airways with associated tertiary lymphoid follicles on immunostained lung sections. Analysis of inflammatory cells in airway walls revealed an influx of CD4+ and CD8+ T cells in both ConB groups and in patients with COPD. In contrast, neutrophils were increased in airways of patients with COPD but not those of patients with ConB (Figures 2A–2C). Compared with ND control subjects, the percentage of small airways with tertiary lymphoid follicles was significantly higher in soldiers with ConB and patients with GOLD stages III and IV COPD and trended higher in patients with sporadic ConB (Figure 2D). Together, these findings suggest that persistent adaptive inflammation in small airways is characteristic of ConB.

Figure 2.

Figure 2.

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Quantification of immune and inflammatory cells in small airways. (A–D) Box-and-whisker plots showing presented CD4+ and CD8+ cells, neutrophils, and tertiary lymphoid follicles. Boxes represent the interquartile range, whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the box, circles beyond the whiskers represent extreme values, and the line within each box represents the median. Groups were compared pairwise using the Mann-Whitney U test. P values were Bonferroni adjusted for multiple comparisons. The threshold for significance was 0.05. Asterisks denote significant differences compared with ND controls (**P < 0.01 and ***P < 0.001), daggers denote significant differences compared with soldiers with constrictive bronchiolitis (ConB) (†††P < 0.001), and double daggers denote significant differences compared with sporadic ConB groups (‡‡‡P < 0.001). CD = cluster of differentiation; COPD = chronic obstructive pulmonary disease; ND = nondiseased.

Small Airway Pathology in ConB Is Distinct from That in Patients with COPD and ND Subjects

To investigate the ability of histomorphometric parameters to separate patients with different diagnoses, we performed PCA on the basis of all small airway morphometric parameters measured for all 138 study participants (Figure 3). This analysis revealed a clear separation of patients with ConB (soldiers and sporadic) from ND control subjects and patients with COPD. In addition, the analysis showed extensive overlap between soldiers with ConB and patients with sporadic ConB, indicating important similarities between the two groups. In contrast, patients with COPD were distinct from ND control subjects and patients with ConB, and patients with GOLD stages III and IV COPD had markedly different airway parameters compared with those with GOLD stages I and II COPD. Further analysis showed that principal component 1 explained diversity among patients on the basis of a mixture of structural (primarily attachment loss and epithelial layer thickness) and inflammatory (neutrophil influx and lymphoid follicles) parameters, resulting in the clear separation of the two groups of patients with COPD. Principal component 2 separated ConB groups from ND control subjects primarily according to CD4+ and CD8+ T-cell accumulation, smooth muscle thickness, and collagen deposition parameters. The relative contributions of individual small airway measurements to the first two PCA dimensions are presented in Table 2.

Figure 3.

Figure 3.

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Principal-component analysis showing separation between groups of patients, with percentages of explained diversity among measured components of small airway immunopathology. ConB = constrictive bronchiolitis; COPD = chronic obstructive pulmonary disease; ND = nondiseased; PC = principal component.

Table 2.

Relative Contributions of Small Airway Histopathological Parameters to Principal Component Analysis

Small Airway Characteristic PC1, % PC2, %
VS(epi,rbm), μm 13.9* 2.8
VS(lp,rbm), μm 8.0 0.0
VS(sm,rbm), μm 0.6 13.4*
VS(adv,rbm), μm 12.2 0.0
Attachments/mm† 15.9* 0.6
Collagen 10.2 5.3*
Elastin 4.0 0.7
Mucus 10.7 0.2
CD8/mm† 1.6 34.0*
CD4/mm† 1.0 33.0*
Neutrophils/mm† 15.8* 0.0
Airways with follicles, % 6.1* 10.1
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Definition of abbreviations: CD = cluster of differentiation; PC = principal component; VS(adv,rbm) = adventitia thickness; VS(epi,rbm) = epithelial height; VS(lp,rbm) = lamina propria thickness; VS(sm,rbm) = smooth muscle layer thickness.

*

Parameters with the greatest contribution to each PC.

†

Values are normalized to 1 mm of reticular basement membrane length.

Lymphocyte Activation Signature in ConB

To further characterize the immune response in patients with ConB, we evaluated the expression of 579 immune-related genes using NanoString gene expression analysis from RNA extracted from lung blocks containing small airways from soldiers with ConB, patients with sporadic ConB, patients with COPD (GOLD stage II), and ND control subjects (six from each group). Differential expression analysis in samples from soldiers with ConB and patients with sporadic ConB compared with ND control subjects showed 88 and 166 differentially expressed genes, respectively, with a change of at least twofold and an adjusted P value of <0.05. Forty-eight genes were commonly upregulated in both ConB groups compared with ND control subjects (see Table E4). In contrast, no genes were found to be differentially expressed between soldiers and patients with sporadic ConB. We then performed cell type profiling using nSolver 4.0 Analysis Software and compared each ConB group with ND control subjects. This analysis showed an increased T-cell score and B-cell score in both ConB groups compared with ND control subjects, confirming the increase in lymphocytes identified by quantification of T cells and lymphoid follicles on immunostained lung sections (Figures 4A and 4B).

Figure 4.

Figure 4.

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Differential expression of immune and inflammatory genes in patients with constrictive bronchiolitis (ConB). (A and B) Box-and-whisker plots showing significantly increased inflammatory T and B cell scores for soldiers with ConB and patients with sporadic ConB compared with nondiseased (ND) control subjects. Inflammatory cell scores were calculated on the basis of gene expression analysis (the threshold for significance was 0.05). Boxes represent the interquartile range, whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the box, and the line within each box represents the median. (C and D) Volcano plots showing differential gene expression from the lungs of soldiers with ConB (C) or patients with sporadic ConB (D) compared with ND control subjects. Inflammatory genes labeled in the plots are commonly upregulated and related to NF-κB, IFN-γ, and TNF signaling. Gray dots represent genes not different from ND control subjects. Green dots represent genes with at least twofold difference and adjusted P values of >0.05. Red dots represent genes with at least twofold difference and adjusted P values of less than 0.05. The x-axes show log2 fold changes in gene expression, and the y-axes show negative log10 of Benjamini-Hochberg–adjusted P values. B2M = beta-2-microglobulin; BCL3 = BCL3 transcription coactivator; C3 = complement C3; CCL = C-C chemokine ligand; CCR = C-C chemokine receptor; CD = cluster of differentiation; CD3D = CD3 delta subunit of T-cell receptor complex; CD3E = CD3 epsilon subunit of T-cell receptor complex; CD40LG = CD40 ligand; CFP = complement factor properdin; CLEC4E = C-type lectin domain family 4 member E; COPD = chronic obstructive pulmonary disease; CXCL = C-X-C motif chemokine ligand; DE = differentially expressed; FKBP5 = FKBP prolyl isomerase 5; GZMA = granzyme A; IFNG = IFN gamma; IL1R2 = IL-1 receptor type 2; IL1RL1 = IL-1 receptor like 1; IL2RB = IL-2 receptor subunit beta; IL2RG = IL-2 receptor subunit gamma; IL18RAP = IL-18 receptor accessory protein; IRF4 = IFN regulatory factor 4; LAMP3 = lysosomal associated membrane protein 3; LILRA5 = leukocyte immunoglobulin like receptor A5; LILRB2 = leukocyte immunoglobulin like receptor B2; LTA = lymphotoxin alpha; MAP4K1 = mitogen-activated protein kinase kinase kinase kinase 1; MS4A1 = membrane spanning 4-domains A1; NF-κB = nuclear factor-κB; RARRES3 = retinoic acid receptor responder 1; S100A8 = S100 calcium binding protein A8; S100A9 = S100 calcium binding protein A9; SLAMF7 = SLAM family member 7; STAT4 = signal transducer and activator of transcription 4; TAP2 = transporter 2, ATP binding cassette subfamily B member; TIGIT = T cell immunoreceptor with Ig and ITIM domains; TNF = tumor necrosis factor; TNFSF13B = TNF superfamily member 13b; XCR1 = X-C motif chemokine receptor 1; ZBTB16 = zinc finger and BTB domain containing 16.

Next, we assessed overrepresented immune and inflammatory pathways in patients with ConB by overlaying differentially expressed genes with Kyoto Encyclopedia of Genes and Genomes pathways using Pathway Score and Path View modules of nSolver 4.0 Analysis software (44). Top pathways enriched in differentially expressed genes were similar in each ConB group compared with ND control subjects (Table 3). In addition to pathways annotated as hematopoietic cell lineage and cell adhesion molecules, which likely reflect increased lymphocyte presence in ConB, other overrepresented pathways point to a persistent lymphocyte activation profile. These pathways included cytokine–cytokine receptor interactions, NF-κB (nuclear factor-κB) signaling, TLR (Toll-like receptor) signaling, T-cell receptor signaling, antigen processing and presentation, and natural killer cell–mediated cytotoxicity. Overexpressed genes in the NF-κB signaling pathway overlap with TLR signaling and cytokine–cytokine receptor interactions and indicate upregulation of both canonical and noncanonical NF-κB pathways (see Figure E3). Differential genes in the cytokine–cytokine receptor pathway (see Figure E4) represent a variety of molecules that are regulated by (and regulate) inflammatory signaling pathways, particularly NF-κB, IFN-γ, and TNF (tumor necrosis factor) signaling. Volcano plots highlighting commonly upregulated genes related to NF-κB, IFN-γ, and TNF signaling in both soldiers with ConB and patients with sporadic ConB are shown in Figures 4C and 4D.

Table 3.

List of Commonly Upregulated Gene Expression Pathways from Lungs of Soldiers with Constrictive Bronchiolitis and Patients with Sporadic Constrictive Bronchiolitis

Common Overrepresented Pathways in ConB  
Cytokine–cytokine receptor interaction  
Cell adhesion molecules  
Hematopoietic cell lineage  
Antigen processing and presentation  
NF-κB signaling pathway  
Natural killer cell–mediated cytotoxicity  
T-cell receptor signaling pathway  
Toll-like receptor signaling pathway  
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Definition of abbreviations: ConB = constrictive bronchiolitis; NF-κB = nuclear factor-κB.

Compared with ND control subjects, lungs from patients with COPD showed 114 differentially expressed genes with at least twofold change in expression and an adjusted P value of <0.05 (Figure 4E). Overrepresented immune and inflammatory pathways in patients with COPD included cytokine–cytokine receptor interactions, NF-κB signaling, and TLR signaling (see Figures E5 and E6). In comparison with lungs from soldiers with ConB, lungs from patients with COPD had only 15 differentially expressed genes, including 11 that were upregulated (LILRB2 [leukocyte immunoglobulin like receptor B2], CFP [complement factor properdin], CD163 [CD163 molecule], LILRA5 [leukocyte immunoglobulin like receptor A5], CLEC4E [C-type lectin domain family 4 member E], BCL3 [BCL3 transcription coactivator], IL18RAP [IL-18 receptor accessory protein], S100A9 [S100 calcium binding protein A9], S100A8 [S100 calcium binding protein A8], IL6, and IL1R2 [IL-1 receptor type 2]). Together, these data indicate that lungs from patients with sporadic ConB and soldiers with ConB share a common immunophenotype dominated by chronic T-cell activation; however, lungs from patients with COPD contain additional markers of innate immune activation.

Discussion

By quantitative morphometric analysis, we found similar pathological abnormalities in small airways of two groups of patients with ConB, including increased thickness of the smooth muscle layer, collagen deposition in the subepithelium, and lymphocyte influx. In contrast, COPD represents a more complex pathology that involves inner, mural, and outer compartments of small airways (45). The pathological abnormalities in ConB are clearly separable from small airway disease associated with COPD. Analysis of inflammatory cells showed a lymphocyte influx in both ConB and COPD, whereas neutrophilic inflammation was present only in patients with COPD. Differential gene expression analysis revealed T-cell activation in the lungs of both groups of patients with ConB compared with control subjects, thereby suggesting that an ongoing adaptive immune response could underlie persistence and progression of pathology in ConB. Compared with lungs from patients with ConB, lungs from patients with COPD shared aspects this immune activation phenotype but also showed upregulation of additional genes involved in innate immunity. Together, these studies highlight similarities among soldiers with ConB and patients with sporadic ConB and suggest a common pathobiology.

Subepithelial collagenous scarring in small airway walls is the histological hallmark of ConB (1, 5, 23, 31, 41, 46, 47). Although not all authors have included smooth muscle hypertrophy or hyperplasia in the definition of ConB (31), our data support the inclusion of both collagen deposition and smooth muscle hypertrophy as common manifestations of ConB. In our study, comparison of small airways from soldiers with ConB and patients with sporadic ConB, who had much more severe clinical disease, showed that patients with sporadic ConB had greater lamina propria thickness, as well as increased thickness of the adventitial layer. In contrast, we found a similar degree of smooth muscle thickness and collagen content (as a percentage of subepithelial area) in both groups. Although increased airway stiffness resulting from collagen deposition likely forms the basis for physiological abnormities in the lung of patients with ConB (48), our findings suggest that progressive extracellular matrix deposition in the lamina propria and adventitia may be important for lung function decline in more severe disease. Our finding that increased collagen content (density) in small airway walls was not associated with lung function decline in patients with ConB is supported by Markopoulo and colleagues, who also showed that the degree of subepithelial collagen deposition itself did not correlate with airflow indices in patients with ConB (46).

Consistent with prior reports, we found that the “itis” in ConB consists of lymphocytes and tertiary lymphoid aggregates (10, 13, 20, 21, 47, 49–57). Our findings are consistent with those of prior studies of ConB and other forms of occupational bronchiolitis, such as silo filler’s disease and flock worker’s lung, in which lymphocytic infiltration, lymphoid follicles, and lymphoid hyperplasia have been reported (10, 20, 21). Both CD4+ and CD8+ T cells are increased in postinfectious childhood ConB biopsy specimens (54, 55). Although the role of lymphocytes in disease onset and progression is uncertain for most forms of ConB, CD8+ T cells have been shown to mediate bronchiolar epithelial damage, and CD4+ T cells contribute to ongoing inflammation and fibrosis in models of bronchiolitis obliterans syndrome (BOS), which is the most common form of chronic lung allograft dysfunction after lung transplantation (47, 49–53). Immune profiling studies in BOS have shown that infiltrating CD4+ and CD8+ T cells release a variety of inflammatory cytokines, especially T-helper cell type 1 (Th1) cytokines such as IFN-γ, IL-2, TNF-α, and IL-4 and IL-6 (13, 54, 56, 57).

Consistent with studies in humans and animal models of BOS (58, 59), we identified a striking Th1 pattern of gene expression in a set of 48 genes commonly upregulated (more than twofold) in the lungs of both patients with sporadic ConB and soldiers with ConB. These genes included a variety of chemokines (CCL3 [C-C chemokine ligand 3], CCL5, CXCL9 [C-X-C motif chemokine ligand 9], CXCL10, CXCL11, and CXCL12), IFN pathway genes (IRF4 [IFN regulatory factor 4]), and TNF-α. In addition to TLR and NF-κB pathways, which control expression of chemokine genes and TNF-α, analysis of all differentially regulated gene products in ConB identified pathways involving cytokine–cytokine receptor interactions, cell adhesion, T-cell receptor signaling, and antigen processing and presentation. Together, these differentially regulated genes and pathways indicate that persistent Th1-type immune activation is a common feature of ConB. In addition, we identified evidence for involvement of CD8+ T cells and B cells in ConB, including upregulation of CD8+ T-cell cytotoxicity genes (GZMA [granzyme A]) and noncanonical NF-κB activation, which is involved in lymphoid tissue homing, B-cell survival, and maturation.

Although Th2 mediators (particularly IL-13) are more commonly associated with fibrotic conditions, Th1 responses appear to contribute to BOS after lung transplantation (13, 59, 60). In this regard, modulation of the Th1/Th17 balance has been shown to prevent disease in an experimental model of BOS, and activation of the CD28/B7-2 pathway regulates development of pathology in mice after tracheal transplantation via promotion of Th1 cytokine expression (58, 61). The specific mechanisms linking Th1 immune activation to airway pathology in humans with ConB require further study.

Our study has some limitations, including the retrospective nature of data collection, the availability of only FFPE lung tissue, and the fact that lungs were not inflated or perfused before fixation. However, we used recommended techniques for quantitative morphometric analysis (42), and all lung samples were processed similarly, thereby reducing the likelihood of bias. Because most lung biopsies in this study were small, it was not possible to determine how widespread the pathological changes were in individual lungs or whether there were regional differences in affected lungs. In this study, patients with ConB were younger than those with COPD, and most were nonsmokers; therefore, we cannot exclude that some of the differences we identified between these groups could be age or smoking related.

Conclusions

We showed that small airway remodeling in ConB is distinct from COPD and consists of collagen deposition, smooth muscle hyperplasia and hypertrophy, and lymphocytic infiltration in the airway wall. Persistent T-cell activation is a common feature of ConB that may account for disease persistence and progression. Together, our data suggest that individualized therapeutic approaches will be necessary to effectively interdict in different forms of small airway disease. Future studies are warranted to better define the natural history of ConB, compare the immune signature in other disease subgroups, and investigate whether immune-targeted therapies could be effective in this disease.

Footnotes

Supported by U.S. Department of Defense grant W81XWH-17-1-0503, the U.S. Department of Veterans Affairs, NIH grant HL126176, and a generous donation from Ms. Carol Odess.

Author Contributions: S.S.G., R.-H.D., and V.V.P. conducted the histopathological studies; J.W.L., L.B.W., and C.M.S. provided human lung samples; S.V.N. and S.S.G. performed gene expression analysis; P.W. and S.S.G. performed statistical analysis; B.W.R. and R.F.M. provided clinical data and performed analysis; J.E.J. performed pathological diagnosis confirmation; J.H.N. and S.I.R. provided scientific and technical knowledge; S.S.G., V.V.P., and T.S.B. wrote the manuscript; and V.V.P., S.I.R., and T.S.B. designed and cosupervised the project. All authors had final approval of the manuscript.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.202109-2133OC on May 13, 2022

Author disclosures are available with the text of this article at www.atsjournals.org.

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