Pirfenidone in fibrotic hypersensitivity pneumonitis: a double-blind, randomised clinical trial of efficacy and safety.

BACKGROUND: Fibrotic hypersensitivity pneumonitis (FHP) is an irreversible lung disease with high morbidity and mortality. We sought to evaluate the safety and effect of pirfenidone on disease progression in such patients. METHODS: We conducted a single-centre, randomised, double-blinded, placebo-controlled trial in adults with FHP and disease progression. Patients were assigned in a 2:1 ratio to receive either oral pirfenidone (2403 mg/day) or placebo for 52 weeks. The primary end point was the mean absolute change in the per cent predicted forced vital capacity (FVC%). Secondary end points included progression-free survival (PFS, time to a relative decline ≥10% in FVC and/or diffusing capacity of the lung for carbon monoxide (DLCO), acute respiratory exacerbation, a decrease of ≥50 m in the 6 min walk distance, increase or introduction of immunosuppressive drugs or death), change in FVC slope and mean DLCO%, hospitalisations, radiological progression of lung fibrosis and safety. RESULTS: After randomising 40 patients, enrolment was interrupted by the COVID-19 pandemic. There was no significant between-group difference in FVC% at week 52 (mean difference -0.76%, 95% CI -6.34 to 4.82). Pirfenidone resulted in a lower rate of decline in the adjusted FVC% at week 26 and improved PFS (HR 0.26, 95% CI 0.12 to 0.60). Results for other secondary end points showed no significant difference between groups. No deaths occurred in the pirfenidone group and one death (respiratory) occurred in the placebo group. There were no treatment-emergent serious adverse events. CONCLUSIONS: The trial was underpowered to detect a difference in the primary end point. Pirfenidone was found to be safe and improved PFS in patients with FHP. TRIAL REGISTRATION MUMBER: NCT02958917.

Lymphangioleiomyomatosis and Other Cystic Lung Diseases.

Cysts and cavities in the lung are commonly encountered on chest imaging. It is necessary to distinguish thin-walled lung cysts (≤2 mm) from cavities and characterize their distribution as focal or multifocal versus diffuse. Focal cavitary lesions are often caused by inflammatory, infectious, or neoplastic processes in contrast to diffuse cystic lung diseases. An algorithmic approach to diffuse cystic lung disease can help narrow the differential diagnosis, and additional testing such as skin biopsy, serum biomarkers, and genetic testing can be confirmatory. An accurate diagnosis is essential for the management and disease surveillance of extrapulmonary complications.

Characterization of Immune Cells From the Lungs of Patients With Chronic Non-Tuberculous Mycobacteria or Pseudomonas aeruginosa Infection.

Little is known of the lung cellular immunophenotypes in patients with non-tuberculous mycobacterial lung disease (NTM-LD). Flow-cytometric analyses for the major myeloid and lymphoid cell subsets were performed in less- and more-diseased areas of surgically resected lungs from six patients with NTM-LD and two with Pseudomonas aeruginosa lung disease (PsA-LD). Lymphocytes, comprised mainly of NK cells, CD4+ and CD8+ T cells, and B cells, accounted for ~60% of all leukocytes, with greater prevalence of T and B cells in more-diseased areas. In contrast, fewer neutrophils were found with decreased number in more-diseased areas. Compared to NTM-LD, lung tissues from patients with PsA-LD demonstrated relatively lower numbers of T and B lymphocytes but similar numbers of NK cells. While this study demonstrated a large influx of lymphocytes into the lungs of patients with chronic NTM-LD, further analyses of their phenotypes are necessary to determine the significance of these findings.

SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted SARS-CoV-2 strain MA10 produces an acute respiratory distress syndrome in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days after virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of profibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early antifibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Design and rationale of a randomised, double-blind trial of the efficacy and safety of pirfenidone in patients with fibrotic hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an immunologically mediated form of lung disease resulting from inhalational exposure to any of a large variety of antigens. A subgroup of patients with HP develops pulmonary fibrosis (fibrotic HP; FHP), a significant cause of morbidity and mortality. This study will evaluate the safety and efficacy of the antifibrotic pirfenidone in treating FHP. This single-centre, randomised, double-blind, placebo-controlled trial is enrolling adults with FHP (ClinicalTrials.gov: NCT02958917). Study participants must have fibrotic abnormalities involving ≥5% of the lung parenchyma on high-resolution computed tomography scan, forced vital capacity (FVC) ≥40% and diffusing capacity of the lung for carbon monoxide ≥30% of predicted values. Study participants will be randomised in a 2:1 ratio to receive pirfenidone 2403 mg·day-1 or placebo. The primary efficacy end-point is the mean change in FVC % predicted from baseline to week 52. A number of secondary end-points have been chosen to evaluate the safety and efficacy in different domains.

Utility of a Molecular Classifier as a Complement to High-Resolution Computed Tomography to Identify Usual Interstitial Pneumonia.

Rationale: Usual interstitial pneumonia (UIP) is the defining morphology of idiopathic pulmonary fibrosis (IPF). Guidelines for IPF diagnosis conditionally recommend surgical lung biopsy for histopathology diagnosis of UIP when radiology and clinical context are not definitive. A "molecular diagnosis of UIP" in transbronchial lung biopsy, the Envisia Genomic Classifier, accurately predicted histopathologic UIP.Objectives: We evaluated the combined accuracy of the Envisia Genomic Classifier and local radiology in the detection of UIP pattern.Methods: Ninety-six patients who had diagnostic lung pathology as well as a transbronchial lung biopsy for molecular testing with Envisia Genomic Classifier were included in this analysis. The classifier results were scored against reference pathology. UIP identified on high-resolution computed tomography (HRCT) as documented by features in local radiologists' reports was compared with histopathology.Measurements and Main Results: In 96 patients, the Envisia Classifier achieved a specificity of 92.1% (confidence interval [CI],78.6-98.3%) and a sensitivity of 60.3% (CI, 46.6-73.0%) for histology-proven UIP pattern. Local radiologists identified UIP in 18 of 53 patients with UIP histopathology, with a sensitivity of 34.0% (CI, 21.5-48.3%) and a specificity of 96.9% (CI, 83.8-100%). In conjunction with HRCT patterns of UIP, the Envisia Classifier results identified 24 additional patients with UIP (sensitivity 79.2%; specificity 90.6%).Conclusions: In 96 patients with suspected interstitial lung disease, the Envisia Genomic Classifier identified UIP regardless of HRCT pattern. These results suggest that recognition of a UIP pattern by the Envisia Genomic Classifier combined with HRCT and clinical factors in a multidisciplinary discussion may assist clinicians in making an interstitial lung disease (especially IPF) diagnosis without the need for a surgical lung biopsy.

Protein tyrosine phosphatase-α amplifies transforming growth factor-ß-dependent profibrotic signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive, often fatal, fibrosing lung disease for which treatment remains suboptimal. Fibrogenic cytokines, including transforming growth factor-ß (TGF-ß), are central to its pathogenesis. Protein tyrosine phosphatase-α (PTPα) has emerged as a key regulator of fibrogenic signaling in fibroblasts. We have reported that mice globally deficient in PTPα (Ptpra-/-) were protected from experimental pulmonary fibrosis, in part via alterations in TGF-ß signaling. The goal of this study was to determine the lung cell types and mechanisms by which PTPα controls fibrogenic pathways and whether these pathways are relevant to human disease. Immunohistochemical analysis of lungs from patients with IPF revealed that PTPα was highly expressed by mesenchymal cells in fibroblastic foci and by airway and alveolar epithelial cells. To determine whether PTPα promotes profibrotic signaling pathways in lung fibroblasts and/or epithelial cells, we generated mice with conditional (floxed) Ptpra alleles (Ptpraf/f). These mice were crossed with Dermo1-Cre or with Sftpc-CreERT2 mice to delete Ptpra in mesenchymal cells and alveolar type II cells, respectively. Dermo1-Cre/Ptpraf/f mice were protected from bleomycin-induced pulmonary fibrosis, whereas Sftpc-CreERT2/Ptpraf/f mice developed pulmonary fibrosis equivalent to controls. Both canonical and noncanonical TGF-ß signaling and downstream TGF-ß-induced fibrogenic responses were attenuated in isolated Ptpra-/- compared with wild-type fibroblasts. Furthermore, TGF-ß-induced tyrosine phosphorylation of TGF-ß type II receptor and of PTPα were attenuated in Ptpra-/- compared with wild-type fibroblasts. The phenotype of cells genetically deficient in PTPα was recapitulated with the use of a Src inhibitor. These findings suggest that PTPα amplifies profibrotic TGF-ß-dependent pathway signaling in lung fibroblasts.

Clinical Decision-Making in Hypersensitivity Pneumonitis: Diagnosis and Management.

This review provides an updated approach to the diagnosis and management of hypersensitivity pneumonitis (HP). The importance of using a multidisciplinary discussion to increase diagnostic and treatment confidence is emphasized. The role of Bayesian reasoning is highlighted throughout, underscoring the importance of hypothesis generation (differential diagnosis) and diagnostic test interpretation based on the probability of HP. Probability estimates of diagnostic certainty (i.e., a confident versus a working diagnosis) and treatment thresholds are carefully examined.Therapeutically, beyond antigen avoidance and newly available antifibrotic therapy for patients with a progressive fibrosing phenotype; the role, timing, and expected response to anti-inflammatory therapy in individual patients are unanswered questions. Since the evidence and validation of testing generally performed during the diagnostic work-up and longitudinal monitoring of HP is feeble at best, the viewpoints discussed are not intended to resolve current controversies but rather to provide a conceptual framework for evaluating discordant information when evaluating and caring for patients with HP.

Use of a molecular classifier to identify usual interstitial pneumonia in conventional transbronchial lung biopsy samples: a prospective validation study.

BACKGROUND: In the appropriate clinical setting, the diagnosis of idiopathic pulmonary fibrosis (IPF) requires a pattern of usual interstitial pneumonia to be present on high-resolution chest CT (HRCT) or surgical lung biopsy. A molecular usual interstitial pneumonia signature can be identified by a machine learning algorithm in less-invasive transbronchial lung biopsy samples. We report prospective findings for the clinical validity and utility of this molecular test. METHODS: We prospectively recruited 237 patients for this study from those enrolled in the Bronchial Sample Collection for a Novel Genomic Test (BRAVE) study in 29 US and European sites. Patients were undergoing evaluation for interstitial lung disease and had had samples obtained by clinically indicated surgical or transbronchial biopsy or cryobiopsy for pathology. Histopathological diagnoses were made by experienced pathologists. Available HRCT scans were reviewed centrally. Three to five transbronchial lung biopsy samples were collected from all patients specifically for this study, pooled by patient, and extracted for transcriptomic sequencing. After exclusions, diagnostic histopathology and RNA sequence data from 90 patients were used to train a machine learning algorithm (Envisia Genomic Classifier, Veracyte, San Francisco, CA, USA) to identify a usual interstitial pneumonia pattern. The primary study endpoint was validation of the classifier in 49 patients by comparison with diagnostic histopathology. To assess clinical utility, we compared the agreement and confidence level of diagnosis made by central multidisciplinary teams based on anonymised clinical information and radiology results plus either molecular classifier or histopathology results. FINDINGS: The classifier identified usual interstitial pneumonia in transbronchial lung biopsy samples from 49 patients with 88% specificity (95% CI 70-98) and 70% sensitivity (47-87). Among 42 of these patients who had possible or inconsistent usual interstitial pneumonia on HRCT, the classifier showed 81% positive predictive value (95% CI 54-96) for underlying biopsy-proven usual interstitial pneumonia. In the clinical utility analysis, we found 86% agreement (95% CI 78-92) between clinical diagnoses using classifier results and those using histopathology data. Diagnostic confidence was improved by the molecular classifier results compared with histopathology results in 18 with IPF diagnoses (proportion of diagnoses that were confident or provisional with high confidence 89% vs 56%, p=0·0339) and in all 48 patients with non-diagnostic pathology or non-classifiable fibrosis histopathology (63% vs 42%, p=0·0412). INTERPRETATION: The molecular test provided an objective method to aid clinicians and multidisciplinary teams in ascertaining a diagnosis of IPF, particularly for patients without a clear radiological diagnosis, in samples that can be obtained by a less invasive method. Further prospective clinical validation and utility studies are planned. FUNDING: Veracyte.

Pulmonary Manifestations of Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) is associated with significant chronic lung disease. The purpose of this paper was to describe the clinical, radiologic, and pathologic findings of CVID-associated lung diseases. These include airways' disease, interstitial lung disease, lymphoma, and mucosa-associated lymphoid tissue lymphoma. In addition, a genetic syndrome termed Kabuki syndrome results in CVID-like immune abnormalities. These patients may also present with CVID-associated lung disease. Awareness and precise identification of CVID-associated lung disease may allow for better assessment of prognosis and direction of therapy.

Protein Tyrosine Phosphatase-N13 Promotes Myofibroblast Resistance to Apoptosis in Idiopathic Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic interstitial lung disease characterized by (myo)fibroblast accumulation and collagen deposition. Resistance to Fas-induced apoptosis is thought to facilitate (myo)fibroblast persistence in fibrotic lung tissues by poorly understood mechanisms. OBJECTIVES: To test the hypothesis that PTPN13 (protein tyrosine phosphatase-N13) is expressed by IPF lung (myo)fibroblasts, promotes their resistance to Fas-induced apoptosis, and contributes to the development of pulmonary fibrosis. METHODS: PTPN13 was localized in lung tissues from patients with IPF and control subjects by immunohistochemical staining. Inhibition of PTPN13 function in primary IPF and normal lung (myo)fibroblasts was accomplished by: 1) downregulation with TNF-α (tumor necrosis factor-α)/IFN-γ, 2) siRNA knockdown, or 3) a cell-permeable Fas/PTPN13 interaction inhibitory peptide. The role of PTPN13 in the development of pulmonary fibrosis was assessed in mice with genetic deficiency of PTP-BL, the murine ortholog of PTPN13. MEASUREMENTS AND MAIN RESULTS: PTPN13 was constitutively expressed by (myo)fibroblasts in the fibroblastic foci of patients with IPF. Human lung (myo)fibroblasts, which are resistant to Fas-induced apoptosis, basally expressed PTPN13 in vitro. TNF-α/IFN-γ or siRNA-mediated PTPN13 downregulation and peptide-mediated inhibition of the Fas/PTPN13 interaction in human lung (myo)fibroblasts promoted Fas-induced apoptosis. Bleomycin-challenged PTP-BL-/- mice, while developing inflammatory lung injury, exhibited reduced pulmonary fibrosis compared with wild-type mice. CONCLUSIONS: These findings suggest that PTPN13 mediates the resistance of human lung (myo)fibroblasts to Fas-induced apoptosis and promotes pulmonary fibrosis in mice. Our results suggest that strategies aimed at interfering with PTPN13 expression or function may represent a novel strategy to reduce fibrosis in IPF.

The relationship between complement C3 expression and the MUC5B genotype in pulmonary fibrosis.

The common gain-of-function MUC5B promoter variant ( rs35705950 ) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31-1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15-2.20); P < 0.05; TT vs. GT = 1.66-fold (95% CI 1.20-2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23- 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF.

Photodynamic therapy via navigational bronchoscopy for peripheral lung cancer in dogs.

OBJECTIVE: In the setting of lung cancer, photodynamic therapy (PDT) is typically used to treat centrally located endobronchial tumors. The development of navigational bronchoscopy has opened the potential for using PDT to treat peripheral lung tumors. However, there is limited information about the feasibility of this approach for treating peripheral lung cancers, and about its effects on surrounding healthy lung tissue. We studied the use of PDT delivered by electromagnetic navigational bronchoscopy to treat peripheral lung cancer in dogs. MATERIALS AND METHODS: Three dogs with peripheral lung adenocarcinomas were given intravenous porfimer sodium (Photofrin® [Pinnacle Biologics, Inc., Chicago, IL]) to photosensitize the tumors, then navigational bronchoscopy was used to deliver photoradiation. One week after PDT, the tumors and involved lung lobe were surgically excised and evaluated histologically. RESULTS: PDT was successful in all three dogs and was associated with tolerable and manageable adverse effects. Tissue sections from within PDT-treated tumors showed regions of coagulative central necrosis admixed with small numbers of inflammatory cells, and arterial thrombosis. Viable adenocarcinoma was seen in the surrounding areas. CONCLUSION: These results suggest that PDT can be successfully deployed to treat peripheral lung cancers using navigational bronchoscopy. Furthermore, damage to surrounding noncancerous tissues can be minimized with accurate placement of the optical fiber. Studies of this modality to treat peripheral lung cancers in humans may be warranted. Lasers Surg. Med. 50:483-490, 2018. © 2018 Wiley Periodicals, Inc.

Usual Interstitial Pneumonia Can Be Detected in Transbronchial Biopsies Using Machine Learning.

RATIONALE: Usual interstitial pneumonia (UIP) is the histopathologic hallmark of idiopathic pulmonary fibrosis. Although UIP can be detected by high-resolution computed tomography of the chest, the results are frequently inconclusive, and pathology from transbronchial biopsy (TBB) has poor sensitivity. Surgical lung biopsy may be necessary for a definitive diagnosis. OBJECTIVES: To develop a genomic classifier in tissue obtained by TBB that distinguishes UIP from non-UIP, trained against central pathology as the reference standard. METHODS: Exome enriched RNA sequencing was performed on 283 TBBs from 84 subjects. Machine learning was used to train an algorithm with high rule-in (specificity) performance using specimens from 53 subjects. Performance was evaluated by cross-validation and on an independent test set of specimens from 31 subjects. We explored the feasibility of a single molecular test per subject by combining multiple TBBs from upper and lower lobes. To address whether classifier accuracy depends upon adequate alveolar sampling, we tested for correlation between classifier accuracy and expression of alveolar-specific genes. RESULTS: The top-performing algorithm distinguishes UIP from non-UIP conditions in single TBB samples with an area under the receiver operator characteristic curve (AUC) of 0.86, with specificity of 86% (confidence interval = 71-95%) and sensitivity of 63% (confidence interval = 51-74%) (31 test subjects). Performance improves to an AUC of 0.92 when three to five TBB samples per subject are combined at the RNA level for testing. Although we observed a wide range of type I and II alveolar-specific gene expression in TBBs, expression of these transcripts did not correlate with classifier accuracy. CONCLUSIONS: We demonstrate proof of principle that genomic analysis and machine learning improves the utility of TBB for the diagnosis of UIP, with greater sensitivity and specificity than pathology in TBB alone. Combining multiple individual subject samples results in increased test accuracy over single sample testing. This approach requires validation in an independent cohort of subjects before application in the clinic.

Genome-wide imputation study identifies novel HLA locus for pulmonary fibrosis and potential role for auto-immunity in fibrotic idiopathic interstitial pneumonia.

BACKGROUND: Fibrotic idiopathic interstitial pneumonias (fIIP) are a group of fatal lung diseases with largely unknown etiology and without definitive treatment other than lung transplant to prolong life. There is strong evidence for the importance of both rare and common genetic risk alleles in familial and sporadic disease. We have previously used genome-wide single nucleotide polymorphism data to identify 10 risk loci for fIIP. Here we extend that work to imputed genome-wide genotypes and conduct new RNA sequencing studies of lung tissue to identify and characterize new fIIP risk loci. RESULTS: We performed genome-wide genotype imputation association analyses in 1616 non-Hispanic white (NHW) cases and 4683 NHW controls followed by validation and replication (878 cases, 2017 controls) genotyping and targeted gene expression in lung tissue. Following meta-analysis of the discovery and replication populations, we identified a novel fIIP locus in the HLA region of chromosome 6 (rs7887 P meta = 3.7 × 10(-09)). Imputation of classic HLA alleles identified two in high linkage disequilibrium that are associated with fIIP (DRB1*15:01 P = 1.3 × 10(-7) and DQB1*06:02 P = 6.1 × 10(-8)). Targeted RNA-sequencing of the HLA locus identified 21 genes differentially expressed between fibrotic and control lung tissue (Q < 0.001), many of which are involved in immune and inflammatory response regulation. In addition, the putative risk alleles, DRB1*15:01 and DQB1*06:02, are associated with expression of the DQB1 gene among fIIP cases (Q < 1 × 10(-16)). CONCLUSIONS: We have identified a genome-wide significant association between the HLA region and fIIP. Two HLA alleles are associated with fIIP and affect expression of HLA genes in lung tissue, indicating that the potential genetic risk due to HLA alleles may involve gene regulation in addition to altered protein structure. These studies reveal the importance of the HLA region for risk of fIIP and a basis for the potential etiologic role of auto-immunity in fIIP.

Relationship between gene expression and lung function in Idiopathic Interstitial Pneumonias.

BACKGROUND: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. RESULTS: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. CONCLUSIONS: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.