Uteroglobin Protective Role in CLAD, from Proteomic to In Vitro Biological Activity

Abstract

Purpose The role of Uteroglobin, a Clara cell secretory protein, in the development of CLAD, is controversial. A proteomic survey suggested its protective role, while other studies reported BAL-fluid or serum increase of uteroglobin levels in BOS patients. Aim of present work was to clarify the possible role of UG in lung repair and in fibrogenesis underlying CLAD development. Methods and Materials Bronchoalveolar lavage fluid (BAL-f) samples from 24 patients (coupled pre- and post-CLAD samples) were analyzed to identify the whole proteome by Liquid Chromatography-Mass Spectrometry (LC-MS). All analyses were carried out on a HPLC coupled to ESI-ION Trap Mass Spectrometer (LC-MS, Thermo Finningan, San Jose, CA, USA). Mesenchimal cells (MC) cultures from BAL-f of patients at pre- and post-CLAD stages were equipped to assess UG activity. A specific colorimetric assay was applied to quantify collagen synthesis. ELISA was applied for Cytokine release assay. Results BAL-f analysis by LC-MS confirmed the identification of many proteins and showed a frequent presence of inflammatory cytokines, small proteins and peptides with molecular masses below 10 kDa. UG was detectable only in pre-CLAD samples of 14 patients with slowly progressive and late lung dysfunction while it was lacking in all but one POST CLAD samples and in pre-CLAD fluid of those patients who developed an earlier and rapidly progressive CLAD. Furthermore UG was able to inhibit collagen production by MC obtained from CLAD patients (mean inhibition 37,3 % at 48 hs) while this effect was not detectable on skin derived fibroblasts. UG significantly inhibited IL8 release by LPS stimulated Alveolar macrophages from CLAD patients (LPS treated Macr 1,45±0,26 mcg/ml versus UG + LPS 0,76±0,04 mcg/ml). Conclusions Our comparative analysis of BAL-f proteomic profiles strongly suggest that UG may play a protective role in CLAD pathogenesis. Its role is confirmed by its ability to modulate in vitro collagen production by CLAD derived MC and IL8 release by macrophages.