Research paperAn immunoproteomic approach revealed antigenic proteins enhancing serodiagnosis performance of bird fancier's lung
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.
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