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Dying blood mononuclear cell secretome exerts antimicrobial activity.

ABSTRACT: BACKGROUND:Several activities are attributed to antimicrobial peptides (AMPs), including bacterial killing, leucocyte recruitment and angiogenesis. Despite promises of advanced cellular therapies for treatment of diabetic foot ulcer, it is currently accepted that paracrine factors rather than cellular components are causative for the observed effects. Whether AMPs are present in the mononuclear cell (MNC) secretome (MNC-sec) of white blood cells that are beneficial in experimental wound healing is not known. MATERIALS AND METHODS:Antimicrobial activity of the secretomes of nonirradiated (MNC-sec) and ?-irradiated MNCs (MNC-sec rad) was analysed by microdilution assay. AMPs were determined by quantitative real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Whether human MNC-sec rad causes AMP secretion in vivo was examined in an experimental rat model. Image flow cytometry was used to determine the type of cell death induced in MNCs after exposure to ?-radiation. RESULTS:The antimicrobial activity assay revealed a bactericidal activity of MNC-sec rad and to a lesser degree also of MNC-sec. Image flow cytometry showed that ?-irradiation of MNCs induced early apoptosis followed mainly by necroptosis. RT-PCR and ELISA revealed a high abundance of different AMPs in the secretome of MNCs. In addition, human MNC-sec elicited an increase in de novo endogenous AMP production in rats in vivo. CONCLUSION:We provide evidence that the secretome of MNCs has direct and indirect positive effects on the immune defence system, including augmentation of antibacterial properties. Our data further suggest that necroptosis could play a key role for the release of paracrine factors and the therapeutic action of MNC-sec rad.

SUBMITTER: Kasiri MM

PROVIDER: S-EPMC5113772 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Dying blood mononuclear cell secretome exerts antimicrobial activity.

Kasiri Mohammad Mahdi MM Beer Lucian L Nemec Lucas L Gruber Florian F Pietkiewicz Sabine S Haider Thomas T Simader Elisabeth Maria EM Traxler Denise D Schweiger Thomas T Janik Stefan S Taghavi Shahrokh S Gabriel Christian C Mildner Michael M Ankersmit Hendrik Jan HJ

European journal of clinical investigation 20160926 10

<h4>Background</h4>Several activities are attributed to antimicrobial peptides (AMPs), including bacterial killing, leucocyte recruitment and angiogenesis. Despite promises of advanced cellular therapies for treatment of diabetic foot ulcer, it is currently accepted that paracrine factors rather than cellular components are causative for the observed effects. Whether AMPs are present in the mononuclear cell (MNC) secretome (MNC-sec) of white blood cells that are beneficial in experimental wound ...[more]

PMID: 27513763

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Project description:BackgroundIgE-mediated hypersensitivity is becoming increasingly prevalent and activation of mast cells and basophils represent key events in the pathophysiology of allergy. We have previously reported that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) exerts beneficial anti-inflammatory effects. Yet, its ability to alleviate allergic symptoms has not been investigated so far.MethodsSeveral experimental in vitro and in vivo models have been used in this basic research study. A murine ear swelling model was used to study the effects of PBMCsec on 48/80-induced mast cell degranulation in vivo. The transcriptional profile of murine mast cells was analysed by single cell RNA sequencing (scRNAseq). Mast cell activation was studied in vitro using primary skin mast cells. Basophils from individuals allergic to birch pollens were used to investigate basophile activation by allergens. Transcriptomic and lipidomic analyses were used to identify mRNA expression and lipid species present in PBMCsec, respectively.FindingsTopical application of PBMCsec on mouse ears (C57BL/6) significantly reduced tissue swelling following intradermal injection of compound 48/80, an inducer of mast cell degranulation. Single cell RNA sequencing of PBMCsec-treated murine dermal mast cells (Balb/c) revealed a downregulation of genes involved in immune cell degranulation and Fc-receptor signalling. In addition, treatment of primary human dermal mast cells with PBMCsec strongly inhibited compound 48/80- and α-IgE-induced mediator release in vitro. Furthermore, PBMCsec remarkably attenuated allergen driven activation of basophils from allergic individuals. Transcriptomic analysis of these basophils showed that PBMCsec downregulated a distinct gene battery involved in immune cell degranulation and Fc-receptor signalling, corroborating results obtained from dermal mast cells. Finally, we identified the lipid fraction of PBMCsec as the major active ingredient involved in effector cell inhibition.InterpretationCollectively, our data demonstrate that PBMCsec is able to reduce activation of mast cells and basophils, encouraging further studies on the potential use of PBMCsec for treating allergy.FundingAustrian Research Promotion Agency (852748 and 862068, 2015-2019), Vienna Business Agency (2343727, 2018-2020), Aposcience AG, Austrian Federal Ministry of Education, Science and Research (SPA06/055), Danube Allergy Research Cluster, Austrian Science Fund (I4437 and P32953).

Project description:Reconstructive surgery transfers viable tissue to cover defects and to restore aesthetic and functional properties. Failure rates after free flap surgery range from 3 to 7%. Co-morbidities such as diabetes mellitus or peripheral vascular disease increase the risk of flap failure up to 4.5-fold. Experimental therapeutic concepts commonly use a monocausal approach by applying single growth factors. The secretome of ?-irradiated, stressed peripheral blood mononuclear cells (PBMCsec) resembles the physiological environment necessary for tissue regeneration. Its application led to improved wound healing rates and a two-fold increase in blood vessel counts in previous animal models. We hypothesized that PBMCsec has beneficial effects on the survival of compromised flap tissue by reducing the necrosis rate and increasing angiogenesis. Surgery was performed on 39 male Sprague-Dawley rats (control, N =?13; fibrin sealant, N =?14; PBMCsec, N =?12). PBMCsec was produced according to good manufacturing practices (GMP) guidelines and 2 ml were administered intraoperatively at a concentration of 2.5?×?107 cells/ml using fibrin sealant as carrier substance. Flap perfusion and necrosis (as percentage of the total flap area) were analyzed using Laser Doppler Imaging and digital image planimetry on postoperative days 3 and 7. Immunohistochemical stainings for von Willebrand factor (vWF) and Vascular Endothelial Growth Factor-receptor-3 (Flt-4) were performed on postoperative day 7 to evaluate formation of blood vessels and lymphatic vessels. Seroma formation was quantified using a syringe and flap adhesion and tissue edema were evaluated clinically through a cranial incision by a blinded observer according to previously described criteria on postoperative day 7. We found a significantly reduced tissue necrosis rate (control: 27.8%?±?8.6; fibrin: 22.0%?±?6.2; 20.9% reduction, p =?.053 vs. control; PBMCsec: 19.1%?±?7.2; 31.1% reduction, p =?.012 vs. control; 12.9% reduction, 0.293 vs. fibrin) together with increased vWF+ vessel counts (control: 70.3 ±?16.3 vessels/4 fields at 200× magnification; fibrin: 67.8 ±?12.1; 3.6% reduction, p =?.651, vs. control; PBMCsec: 85.9 ±?20.4; 22.2% increase, p =?.045 vs. control; 26.7% increase, p =?.010 vs. fibrin) on postoperative day 7 after treatment with PBMCsec. Seroma formation was decreased after treatment with fibrin sealant with or without the addition of PBMCsec. (control: 11.9 ±?9.7 ml; fibrin: 1.7 ±?5.3, 86.0% reduction, 0.004 vs. control; PBMCsec: 0.6 ±?2.0; 94.8% reduction, p =?.001 vs. control; 62.8% reduction, p =?.523 vs. fibrin). We describe the beneficial effects of a secretome derived from ?-irradiated PBMCs on tissue survival, angiogenesis, and clinical parameters after flap surgery in a rodent epigastric flap model.

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Dying blood mononuclear cell secretome exerts antimicrobial activity.

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Dying blood mononuclear cell secretome exerts antimicrobial activity.

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