Elsevier

Journal of Immunological Methods

Volume 450, November 2017, Pages 58-65
Journal of Immunological Methods

Research paper
An immunoproteomic approach revealed antigenic proteins enhancing serodiagnosis performance of bird fancier's lung

https://doi.org/10.1016/j.jim.2017.07.012Get rights and content

Highlights

  • Droppings and blooms contain 14 antigenic proteins.

  • These proteins are involved in either the digestive or immune systems of pigeons.

  • ELISA tests with recombinant IGLL1 and ProE improve and standardize serological diagnosis of BFL.

Abstract

Background

Bird fancier's lung (BFL) caused by repeated inhalation of avian proteins is the most common form of hypersensitivity pneumonitis. However, the exact identification of proteins involved is unknown, and serological test use for diagnosis need to be standardized. The objectives of this study were (i) to identify antigenic proteins from pigeon droppings (ii) to provide information about their location in avian matrices and (iii) to produce them in recombinant proteins to evaluate their diagnostic performances.

Method

Antigenic proteins of pigeon dropping extracts were investigated using 2-dimensional immunoblotting with sera from patients with BFL, asymptomatic exposed controls and healthy volunteers. We investigated the origin of these antigenic proteins by analyzing droppings, blooms and sera using a shotgun proteomic analysis. BFL-associated proteins were produced as recombinant antigens in E. coli and were assessed in ELISA with sera from patients (n = 25) and subject exposed controls (n = 30). These diagnostic performances were compared with those obtained by precipitin techniques (agar gel double diffusion, immunoelectrophoresis).

Results

We identified 14 antigenic proteins mainly located in droppings and blooms. These proteins were involved in either the digestive or immune systems of pigeons.

Using the recombinant BFL-associated proteins: Immunoglobulin lambda-like polypeptide-1 (IGLL1: sensitivity: 76%; specificity: 100%; AUC: 0.93) and Proproteinase E (ProE: sensitivity: 84%; specificity: 80%; AUC: 0.85), the ELISA test showed better performance than precipitin assays with pigeon dropping extracts (sensitivity: 60%; specificity: 93.3%; AUC: 0.76).

Conclusion

IGLL1 and ProE were identified as the biomarkers of the disease. The use of these highly standardized antigens discriminates BFL cases from exposed subjects in serological assays. The results of this study offer new possibilities for the serological diagnosis of the disease.

Clinical trial registration: ClinicalTrials.gov: Identifier NCT03056404.

Introduction

Hypersensitivity pneumonitis (HP) is a group of inflammatory interstitial lung diseases caused by an exaggerated immune response to the inhalation of antigenic particles from environmental sources (Costabel et al., 2012). More than 300 agents have been reported to be involved in HP including bacteria, fungi, animal proteins and chemical compounds (Dalphin and Gondouin, 2015). These antigenic substances can be found in various locations including the home, recreational sites or the workplace. First described by Reed et al. in 1965 (Reed et al., 1965), bird fancier's lung (BFL) is the most common form of HP worldwide with a prevalence ranging from 6% to 20% of exposed pigeon breeders (Costabel et al., 2012).

Regular exposure to avian proteins from droppings, feathers and blooms (white powder that coats the feathers) may cause the disease in sensitized individuals. Following inhalation of antigens, immune complexes are formed in the alveolar and bronchiolar walls leading to tissue damage, fibrosis or emphysema. Evidence is supported by the presence of high titers of specific IgG antibodies to the offending antigens in both the circulation and bronchoalveolar lavage fluids (BAL) (Yoshizawa et al., 1995). The immunologic mechanisms involved in the disease have been described as a combination of type III (immune-complex-mediated reaction) and IV reaction (granuloma formation) with activation of alveolar macrophages and T lymphocytes (Costabel et al., 2012).

The diagnostic procedure of this pathology is complex and requires a combination of clinical, radiological, functional and biological criteria, including the finding of circulating antibodies against the offending antigen. For serological tests, several methods to determine precipitins (immunodiffusion and immunoelectrophoresis) (Grech et al., 2000, Funke and Fellrath, 2008, McSharry et al., 2006) or total IgG antibodies (enzyme-linked immunosorbent assays (ELISA) and ImmunoCAP technique) (Lopata et al., 2004) have been routinely used in analytical laboratories. Some of these techniques were very time-consuming to implement, and the antigens used often lacked standardization.

The efficacy of these tests can be improved by a standardized ELISA using recombinant antigens instead of crude extracts. To move forward in this field, the National Heart, Lung and Blood Institute/Office of Rare Diseases Workshop in Bethesda (Maryland) recommended the validation of biomarkers (exposure and disease) and the development of a battery of standardized antigens that should be available to clinicians for use in diagnosis (Fink et al., 2005). Indeed, there is a lack of knowledge about the precise identity of the antigenic proteins in BFL as well as their location in avian matrices. However, these data are very important both to better understand the disease and to provide breeders with targeted preventive measures. Novel emerging data in Genomics such as the whole genome sequencing of Columba livia (since September 2013) and in Proteomics (LC-MS/MS) now make it possible to characterize new candidate biomarkers for BFL.

Our strategy was to identify by LC-MS/MS all the antigenic proteins revealed by 2D-immunoblotting and also detected on the 2-DE Coomassie blue gel. These proteins were localized by analyzing pigeon droppings, blooms and sera using a shotgun proteomic approach. The antigenic proteins which reacted by immunoblotting with sera from all patients (7/7) and not with sera from control breeders (0/6) were defined as “BFL-associated”. These proteins were produced as recombinant antigens and their diagnostic performances were assessed by ELISA and compared with those of precipitin techniques.

Section snippets

Study population

The protocol was approved by the local ethics committee (CPP-Est II 15/496). Patients with BFL (n = 25) were diagnosed in the Pneumology Unit at the University hospital of Besancon (France) and ULB Erasme Hospital of Brussels (Belgium). All patients were given a diagnosis according to the following criteria (Dalphin and Gondouin, 2015): (i) Exposure to offending antigens, (ii) Symptoms and HRCT compatible with HP and basal crepitant rales, (iii) BAL lymphocytosis, and (iv) Decreased DLCO during

Detection of antigenic proteins by 2D PAGE and immunoblotting

The proteins of droppings were subjected to 2-DE and immunoblotting. The membranes were probed with sera from 7 BFL patients and 6 AECs exclusively exposed to pigeons and 5 HVs to identify BFL-associated spots. First of all, sera from HVs failed to react with any of the proteins (data not shown). The representative 2D-immunoblotting using AECs (Fig. 1A) and BFL (Fig. 1B) sera is shown in Fig. 1. At first sight, the spots displayed on the membrane of the AECs were fewer and less well defined

Discussion

Using an optimal immunoproteomic approach, we have identified by mass spectrometry antigenic proteins which are both related to pigeon exposure and disease-associated. These proteins were involved in either the digestive or immune systems of birds and have been characterized as abundant proteins in droppings and blooms. The IGLL1 and ProE proteins that only reacted with antibodies in all BFL patients and no controls were defined as “BFL-associated” proteins. ProE was the most effective

Conclusion

In pigeon droppings, we found a mixture of antigenic proteins associated with both exposure and the disease. IGLL1 and ProE were BFL-associated proteins located in pigeon droppings and blooms. Using these recombinant antigens in ELISA for routine analysis would provide a more efficient, standardized, reliable and rapid diagnosis than precipitin techniques. This study was part of a continuous effort to improve BFL diagnosis in order to reduce the suffering and stress experienced by patients

Acknowledgments

The authors thank the bird breeders in the Franche-Comté region for their participation, Audrey Laboissière from the Mycology Department of Besancon University Hospital for her excellent technical support, and Dr Ghalia Boubaker from the Parasitology institute of Bern University for her scientific and technical support. The authors are grateful to Pamela Albert for her editorial assistance.

Funding sources

This work was supported by the Besancon University Hospital (N°2015-A01803-46) [API3A (Appel à Projet Interne « 3 axes »), HYPERSENS].

Conflicts of interest

None.

References (39)

  • G. Bestel-Corre et al.

    Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn. by two-dimensional electrophoresis and mass spectrometry

    Electrophoresis

    (2002)
  • G. Boyd et al.

    A current view of pigeon fancier's lung. A model for pulmonary extrinsic allergic alveolitis

    Clin. Allergy

    (1982)
  • R. Craig et al.

    TANDEM: matching proteins with tandem mass spectra

    Bioinformatics

    (2004)
  • J.-C. Dalphin et al.

    Rare Causes and the Spectrum of Hypersensitivity Pneumonitis. Orphan Lung Diseases

    (2015)
  • J.H. Edwards et al.

    Antigens in pigeon breeders' disease

    Immunology

    (1970)
  • J.N. Fink et al.

    Needs and opportunities for research in hypersensitivity pneumonitis

    Am. J. Respir. Crit. Care Med.

    (2005)
  • C. Fu et al.

    Hot fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase

    PLoS One

    (2014)
  • M. Funke et al.

    Hypersensitivity pneumonitis secondary to lovebirds: a new cause of bird fancier's disease

    Eur. Respir. J.

    (2008)
  • F.X. Gomis-Rüth et al.

    The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C

    EMBO J.

    (1995)
  • Cited by (16)

    • Birds of a Feather Precipitate Together

      2022, Archivos de Bronconeumologia
    • Bird fancier's lung serodiagnosis by automated r-IgLL1 ELISA

      2022, Journal of Immunological Methods
      Citation Excerpt :

      Johannson et al., 2020; Bellanger et al., 2019) Recently, the protein immunoglobulin lambda-like polypeptide-1 (IgLL1), isolated from pigeon droppings, was shown to be associated with BFL.( Rouzet et al., 2017a; Shirai et al., 2017) We previously produced this protein as a recombinant antigen and retrospectively assessed its diagnostic performance by a manual ELISA.( Rouzet et al., 2017a) The use of recombinant antigens is recognized to be the best way to avoid variability between batches and laboratories.(

    • A rapid test for the environmental detection of pigeon antigen

      2021, Science of the Total Environment
      Citation Excerpt :

      The immunoblotting results of the present study demonstrated that the polyclonal antibodies used in the methods developed were able to detect the relevant pigeon antigens and that most of the antigenic proteins identified were also abundant in droppings and blooms (Rouzet et al., 2017). Rouzet et al. (2017) found proteins with a molecular weight range between 20 and 200 kD in bird dropping extracts, and that IGLL-1 (Immunoglobulin lambda-like polypeptide-1, 24.5 KDa) and ProE (Proproteinase E, 20.5 KDa) were the antigenic proteins with the most clinical interest as biomarkers for BRHP diagnosis. In this regard, Shirai et al. (2017) in an immunoblot analysis of sera from patients with BRHP, identified multiple immunoreactive bands in pigeon serum at various molecular weights, especially IGLL-1, similar to those obtained in the present study.

    • Diagnosis and Evaluation of Hypersensitivity Pneumonitis: CHEST Guideline and Expert Panel Report

      2021, Chest
      Citation Excerpt :

      Studies are emerging on the use of immunoreactive proteins to circumvent the limitations of standard antigen extract preparation. Presently, such preparations are not yet commercially available or representative of all potential causative antigens of individual HP cases (eg, farmer’s lung).77,90,91 Since the performance characteristics and interpretation of the serum antigen-specific antibody test are often site-specific and influenced by the prevalence of HP in the population being tested (ie, predictive value), the clinician ordering serum antigen-specific antibody testing should understand the validity and reliability of the test and the population sample type and size used to determine its diagnostic performance characteristics.

    View all citing articles on Scopus
    View full text