Background: Invasive aspergillosis is a severe disease caused by the opportunistic mold Aspergillus
which is a major threat for immunocompromised patients such as hematopoietic stem cells transplant
(HSCT) recipients. Neutrophils play an essential role in the control of A. fumigatus infection, as they
are able to phagocyte resting conidia and to act against germinating conidia and hyphae through a
trapping mechanism. We recently found that in HSCT patients, neutrophil-driven immunity against
Aspergillus was altered in relation with calcineurin inhibitor administration (Imbert et al, J Allergy Clin
Immunol 2016). So our work aimed at better understanding the host-pathogen interaction between
neutrophils from HSCT patients and A. fumigatus using a two-dimensional electrophoresis proteomic
approach. This method allows the detection and identification of differentially represented neutrophils
proteins between control and HSCT patient.
Material/methods: Neutrophils from ten blood samples collected from 5 healthy donors and 5 HSCT
patients during the neutrophils recovery (i.e. approximately 15 days after the engraftment) were used.
Two conditions were performed: neutrophils alone or co-cultured with A. fumigatus. After 6 hours of
incubation (37°C, 5% CO2), culture supernatants were collected while neutrophils were lysed. Protein
extract from the different conditions were labeled with different fluorescent dyes (Cy2, Cy3, Cy5) and
submitted to 2D DIGE electrophoresis. Analysis included the detection of the proteins spots in gel by
DeCyder DIA (Difference In-gel Analysis) and a gel-to-gel matching and statistical analyses by
DeCyder BVA (Biological Variation Analysis). Spots of interest were picked and subjected to trypsic
digestion. Resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry
(LC-MS/MS) and Swissprot and TrEMBL databases for protein identification.
Results: We focused on 103 spot that presented significant variations between the different
conditions. Among the tens proteins that we successfully identified, some are presented Table 1.
Conclusions: The differently represented proteins we identified lead to consider that the pathogen
starts up in neutrophil cells a series of reactions that aim to morphological changes, defense
mechanisms against microorganisms and signal to others cells of immune system. Furthermore, we
identified proteins that undergo a change in terms of quantity (after Aspergillus infection) only in
neutrophils from immunocompetent subject. This observation may be linked to the alteration of the
neutrophils response toward Aspergillus in HSCT patients.
Full conference title:
- ECCMID 27th (2017)