July 2020 Paper published in Journal of Allergy and Clinical Immunology
Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic Dermatitis
Rojahn TB1, Vorstandlechner V2, Krausgruber T3, Bauer WM1, Alkon N1, Bangert C1, Thaler FM1, Sadeghyar F1, Fortelny N3, Gernedl V3, Rindler K1, Elbe-Bürger A1, Bock C4, Mildner M1, Brunner PM5
1 Department of Dermatology, Medical University of Vienna, Vienna, Austria.
2 Department of Dermatology, Medical University of Vienna, Vienna, Austria; Department of Surgery, Research Laboratory for Thoracic Surgery, Medical University of Vienna, Vienna, Austria.
3 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.
4 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
5 Department of Dermatology, Medical University of Vienna, Vienna, Austria.
Background: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, but its complex pathogenesis is only insufficiently understood, resulting in still limited treatment options.
Objective: We sought to characterize AD on both transcriptomic and proteomic levels in humans.
Methods: We used skin suction blistering, a painless and nonscarring procedure that can simultaneously sample skin cells and interstitial fluid. We then compared results with conventional biopsies.
Results: Suction blistering captured epidermal and most immune cells equally well as biopsies, except for mast cells and nonmigratory CD163+ macrophages that were only present in biopsy isolates. Using single-cell RNA sequencing, we found comparable transcriptional profiles of key inflammatory pathways between blister and biopsy AD, but suction blistering was superior in cell-specific resolution for high-abundance transcripts (KRT1/KRT10, KRT16/KRT6A, S100A8/S100A9), which showed some background signals in biopsy isolates. Compared with healthy controls, we found characteristic upregulation of AD-typical cytokines such as IL13 and IL22 in Th2 and Th22 cells, respectively, but we also discovered these mediators in proliferating T cells and natural killer T cells, that also expressed the antimicrobial cytokine IL26. Overall, not T cells, but myeloid cells were most strongly enriched in AD, and we found dendritic cell (CLEC7A, amphiregulin/AREG, EREG) and macrophage products (CCL13) among the top upregulated proteins in AD blister fluid proteomic analyses.
Conclusion: These data show that by using cutting-edge technology, suction blistering offers several advantages over conventional biopsies, including better transcriptomic resolution of skin cells, combined with proteomic information from interstitial fluid, unraveling novel inflammatory players that shape the cellular and proteomic microenvironment of AD.
June 2020 Paper published in the International Journal of Molecular Sciences
Role for Lipids Secreted by Irradiated Peripheral Blood Mononuclear Cells in Inflammatory Resolution in Vitro.
Int. J. Mol. Sci. 2020, 21, 4694; doi:10.3390/ijms21134694.
Panahipour L, Kochergina E, Laggner M, Zimmermann M, Mildner M, Ankersmit HJ, Gruber R.
1 Department of Oral Biology, Medical University of Vienna, Sensengasse 2a, 1090 Vienna, Austria;
2 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Währingergürtel 18-20, 1090 Vienna, Austria;
3 Division of Thoracic Surgery, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria
4 Department of Oral and Maxillofacial Surgery, Medical University of Vienna, Währingergürtel 18-20, 1090 Vienna, Austria;
5 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical Universityof Vienna, Währingergürtel 18-20, 1090 Vienna, Austria;
6 Department of Periodontology, School of Dental Medicine, University of Bern, Freiburgstrasse 7, 3010 Bern, Switzerland;
7 Austrian Cluster for Tissue Regeneration, Donaueschingenstraße 13, 1200 Vienna, Austria;
Periodontal inflammation is associated with dying cells that potentially release metabolites helping to promote inflammatory resolution. We had shown earlier that the secretome of irradiated, dying peripheral blood mononuclear cells support in vitro angiogenesis. However, the ability of the secretome to promote inflammatory resolution remains unknown. Here, we determined the expression changes of inflammatory cytokines in murine bone marrow macrophages, RAW264.7 cells, and gingival fibroblasts exposed to the secretome obtained from γ-irradiated peripheral blood mononuclear cells in vitro by RT-PCR and immunoassays. Nuclear translocation of p65 was detected by immunofluorescence staining. Phosphorylation of p65 and degradation of IκB was determined by Western blot. The secretome of irradiated peripheral blood mononuclear cells significantly decreased the expression of IL1 and IL6 in primary macrophages and RAW264.7 cells when exposed to LPS or saliva, and of IL1, IL6, and IL8 in gingival fibroblasts when exposed to IL-1β and TNFα. These changes were associated with decreased phosphorylation and nuclear translocation of p65 but not degradation of IκB in macrophages. We also show that the lipid fraction of the secretome lowered the inflammatory response of macrophages exposed to the inflammatory cues. These results demonstrate that the secretome of irradiated peripheral blood mononuclear cells can lower an in vitro simulated inflammatory response, supporting the overall concept that the secretome of dying cells promotes inflammatory resolution.
June 2020 Paper published in Molecular Therapy
Quantitative Hybrid Cardiac [18F]FDG-PET-MRI Images for Assessment of Cardiac Repair by Preconditioned Cardiosphere-Derived Cells.
Molecular Therapy. Methods & Clinical Development. Vol. 18, 11 Sept 2020, p 354-366. doi: 10.1016/j.omtm.2020.06.008.
Winkler J, Lukovic D, Mester-Tonczar J, Zlabinger K, Gugerell A, Pavo N, Jakab A. Szanka Z, Traxler1 D, Müller C, Spannbauer A, Riesenhuber M, Hasimbegovic E, Dawkins J, Zimmermann M, Ankersmit HJ, Marban E, Gyöngyösi M.
1 Department of Cardiology, Medical University of Vienna, Vienna, Austria
2 Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria
3 Center for MR-Research, University Children’s Hospital Zurich, Steinwiesstrasse 7e, 80cb Zurich, Switzerland
4 Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
5 Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria
Cardiosphere-derived cells (CDCs) are progenitor cells derived from heart tissue and have shown promising results in preclinical models. APOSEC, the secretome of irradiated peripheral blood mononuclear cells, has decreased infarct size in acute and chronic experimental myocardial infarction (MI). We enhanced the effect of CDCs with APOSEC preconditioning (apoCDC) and investigated the reparative effect in a translational pig model of reperfused MI. Supernatants of CDCs, assessed by proteomic analysis, revealed reduced production of extracellular matrix proteins after in vitro APOSEC preconditioning. In a porcine model of catheter-based reperfused anterior acute MI (AMI), CDCs with (apoCDC, n = 8) or without APOSEC preconditioning (CDC, n = 6) were infused intracoronary, 15 min after the start of reperfusion. Untreated AMI animals (n = 7) and sham procedures (n = 5) functioned as controls. 2-deoxy-2-(18 F)-fluoro-D-glucose-positron emission tomography-magnetic resonance imaging ([18F]FDG-PET-MRI), with late enhancement after 1 month, showed reduced scar volume and lower transmurality of the infarcted area in CDC and apoCDC compared to AMI controls. Segmental quantitative PET images displayed indicated more residual viability in apoCDC. The left-ventricle (LV) ejection fraction was improved nonsignificantly to 45.8% ± 8.6% for apoCDC and 43.5% ± 7.1% for CDCs compared to 38.5% ± 4.4% for untreated AMI. Quantitative hybrid [18F]FDG-PET-MRI demonstrated improved metabolic and functional recovery after CDC administration, whereas apoCDCs induced preservation of viability of the infarcted area.
May 2020 Paper published in EBioMedicine
Therapeutic potential of lipids obtained from γ-irradiated PBMCs in dendritic cell-mediated skin inflammation.
EBioMedicine. 2020 May;55:102774. doi: 10.1016/j.ebiom.2020.102774. Epub 2020 May 8.
Maria Laggner 1, Dragan Copic 1, Lucas Nemec 2, Vera Vorstandlechner 1, Alfred Gugerell 1, Florian Gruber 2, Anja Peterbauer 3, Hendrik J Ankersmit 4, Michael Mildner 5
1 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Vienna, Austria; Division of Thoracic Surgery, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
2 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Lazarettgasse 14, 1090 Vienna, Austria.
3 Austrian Red Cross Blood Transfusion Service of Upper Austria, Linz, Austria.
4 Laboratory for Cardiac and Thoracic Diagnosis, Regeneration and Applied Immunology, Vienna, Austria; Division of Thoracic Surgery, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
5 Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Lazarettgasse 14, 1090 Vienna, Austria.
Background: Since numerous pathological conditions are evoked by unwanted dendritic cell (DC) activity, therapeutic agents modulating DC functions are of great medical interest. In regenerative medicine, cellular secretomes have gained increasing attention and valuable immunomodulatory properties have been attributed to the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs). Potential effects of the PBMC secretome (PBMCsec) on key DC functions have not been elucidated so far.
Methods: We used a hapten-mediated murine model of contact hypersensitivity (CH) to study the effects of PBMCsec on DCs in vivo. Effects of PBMCsec on human DCs were investigated in monocyte-derived DCs (MoDC) and ex vivo skin cultures. DCs were phenotypically characterised by transcriptomics analyses and flow cytometry. DC function was evaluated by cytokine secretion, antigen uptake, PBMC proliferation and T-cell priming.
Findings: PBMCsec significantly alleviated tissue inflammation and cellular infiltration in hapten-sensitized mice. We found that PBMCsec abrogated differentiation of MoDCs, indicated by lower expression of classical DC markers CD1a, CD11c and MHC class II molecules. Furthermore, PBMCsec reduced DC maturation, antigen uptake, lipopolysaccharides-induced cytokine secretion, and DC-mediated immune cell proliferation. Moreover, MoDCs differentiated with PBMCsec displayed diminished ability to prime naïve CD4+T-cells into TH1 and TH2 cells. Furthermore, PBMCsec modulated the phenotype of DCs present in the skin in situ. Mechanistically, we identified lipids as the main biomolecule accountable for the observed immunomodulatory effects.
Interpretation: Together, our data describe DC-modulatory actions of lipids secreted by stressed PBMCs and suggest PBMCsec as a therapeutic option for treatment of DC-mediated inflammatory skin conditions.