ABSTRACT
Increasing number of severe COVID 19 patients develop pulmonary Fibrosis, but the management of this complication is still unclear due to a lack of clinical trials. Aim of this study was to characterize mesenchymal cells (MC) isolated from 10 broncho-alveolar lavage (BAL, at 2 months after discharge) from patients with COVID19 fibrosis (COVID19-f) and to compare them with those isolated from 8 patients with collagen tissue diseaseassociated interstitial fibrosis(CTD-ILD). BAL fluid (BALf) levels of TGFbeta, VEGF, TIMP2, RANTES, IL6, IL8, and PAI1 were assessed by ELISA. Primary MC foci were cultured and expanded in D-MEM +10% FBS, characterized by flow cytometry and osteogenic and adipogenic differentiation. Collagen 1 production (+/-TGF-beta) was tested by WB and mRNA expression. BALf cytokine and GF levels were comparable in the two groups. Efficiency of MC isolation from BAL was 100% in COVID-f compared to 65% in CTD-ILD. MC antigen surface expression of CD105, CD73, CD90 (>90%, respectively), CD45, CD34, CD19 and HLA-DR (<5%, respectively) was comparable. None of MC samples differentiated in adipocytes, while COVID19-f were positive for calcium deposition. COVID19-f MC showed at WB, higher Collagen 1 production with respect to CTD-ILD with TGF-beta stimulation. Our preliminary data suggest MC from COVID19-f share several features with CTD-ILD but might have a higher response to fibrogenic and differentiation signals.
ABSTRACT
Neutrophils (Neu) play a pathogenic role in COVID19 by releasing Neutrophils Extracellular Traps (NETs) or HNE. Being HNE inhibited by a1AT, supplementation of this protein has been proposed. We aim to study a1AT/HNE balance in BALf from ICU admitted COVID19 patients. To assess HNE, a1AT and HNE/a1AT complexes, 33 COVID 19 BALf samples were analysed by means of ELISA or gel-Electrophoresis + Western Blot. Proteins bound to a1AT or HNE were identified by Liquid chromatography-mass spectrometry. NETs release (PMA stimulated Neu +/- a1AT) was analysed by confocal microscopy. Both HNE and a1AT were clearly detectable in BALf at high levels. Contrary to what previously observed in other settings (Bronchiolits obliterans) (Cagnone, M. et al. High Throughput 2019;8(1):5) we couldn't detect any HNE/ a1AT complex in COVID19 even when purified HNE was added to samples (Fig 1a). HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free a1AT to bind to histones. Although this binding was confirmed, a1AT wasn't able to inhibit NETs formation (Fig 1b). Despite the finding of a high burden of free and bound HNE in COVID 19 BALf, the formation of HNE/ a1AT inhibitory complex is prevented. Furthermore, a1AT binds to histones but does not prevent NETs formation and their noxious activity.